Packaging systems for human recombinant adenovirus to be used in gene therapy

ABSTRACT

Presented are ways to address the problem of replication competent adenovirus in adenoviral production for use with, for example, gene therapy. Packaging cells having no overlapping sequences with a selected vector and are suited for large scale production of recombinant adenoviruses. A system for use with the invention produces adenovirus incapable of replicating. The system includes a primary cell containing a nucleic acid based on or derived from adenovirus and an isolated recombinant nucleic acid molecule for transfer into the primary cell. The isolated recombinant nucleic acid molecule is based on or derived from an adenovirus, and further has at least one functional encapsidating signal, and at least one functional Inverted Terminal Repeat. The isolated recombinant nucleic acid molecule lacks overlapping sequences with the nucleic acid of the cell. Otherwise, the overlapping sequences would enable homologous recombination leading to replication competent adenovirus in the primary cell into which the isolated recombinant nucleic acid molecule is to be transferred.

RELATED APPLICATIONS

This patent application is a National Stage application under 35 U.S.C.§371 of International patent Application PCT/NL96/00244 filed on Jun.14, 1996 which itself claims priority from European patent application95201728.3 filed on Jun. 26, 1995 and European patent application95201611.1 filed on Jun. 15, 1995.

TECHNICAL FIELD

The invention relates to the field of recombinant DNA technology, morein particular to the field of gene therapy. In particular the inventionrelates to gene therapy using materials derived from adenovirus, inparticular human recombinant adenovirus. It especially relates to novelvirus derived vectors and novel packaging cell lines for vectors basedon adenoviruses.

BACKGROUND

Gene therapy is a recently developed concept for which a wide range ofapplications can be and have been envisaged.

In gene therapy a molecule carrying genetic information is introducedinto some or all cells of a host, as a result of which the geneticinformation is added to the host in a functional format.

The genetic information added may be a gene or a derivative of a gene,such as a cDNA, which encodes a protein. In this case the functionalformat means that protein can be expressed by the machinery of the hostcell.

The genetic information can also be a sequence of nucleotidescomplementary to a sequence of nucleotides (be it DNA or RNA) present inthe host cell. The functional format in this case is that the added DNA(nucleic acid) molecule or copies made thereof in situ are capable ofbase pairing with the complementary sequence present in the host cell.

Applications include the treatment of genetic disorders by supplementinga protein or other substance which is, through said genetic disorder,not present or at least present in insufficient amounts in the host, thetreatment of tumors and (other) acquired diseases such as (auto)immunediseases or infections, etc.

As may be clear from the above, there are basically three differentapproaches in gene therapy, one directed towards compensating adeficiency present in a (mammalian) host; the second directed towardsthe removal or elimination of unwanted substances (organisms or cells)and the third towards application of a recombinant vaccine (tumors orforeign micro-organisms).

For the purpose of gene therapy, adenoviruses carrying deletions havebeen proposed as suitable vehicle. Adenoviruses are non-enveloped DNAviruses. Gene-transfer vectors derived from adenoviruses (so-calledadenoviral vectors) have a number of features that make themparticularly useful for gene transfer for such purposes. Eg. the biologyof the adenoviruses is characterized in detail, the adenovirus is notassociated with severe human pathology, the virus is extremely efficientin introducing its DNA into the host cell, the virus can infect a widevariety of cells and has a broad host-range, the virus can be producedin large quantities with relative ease, and the virus can be renderedreplication defective by deletions in the early-region 1 (E1) of theviral genome.

The adenovirus genome is a linear double-stranded DNA molecule ofapproximately 36000 base pairs with the 55-kDa terminal proteincovalently bound to the 5'terminus of each strand. The Ad DNA containsidentical Inverted Terminal Repeats (ITR) of about 100 base pairs withthe exact length depending on the serotype. The viral origins ofreplication are located within the ITRs exactly at the genome ends. DNAsynthesis occurs in two stages. First, the replication proceeds bystrand displacement, generating a daughter duplex molecule and aparental displaced strand. The displaced strand is single stranded andcan form a so-called "panhandle" intermediate, which allows replicationinitiation and generation of a daughter duplex molecule. Alternatively,replication may proceed from both ends of the genome simultaneously,obviating the requirement to form the panhandle structure. Thereplication is summarized in FIG. 14 adapted from (Lechner and Kelly,1977).

During the productive infection cycle, the viral genes are expressed intwo phases: the early phase, which is the period upto viral DNAreplication, and the late phase, which coincides with the initiation ofviral DNA replication. During the early phase only the early geneproducts, encoded by regions E1, E2, E3 and E4, are expressed, whichcarry out a number of functions that prepare the cell for synthesis ofviral structural proteins (Berk, 1986). During the late phase the lateviral gene products are expressed in addition to the early gene productsand host cell DNA and protein synthesis are shut off. Consequently, thecell becomes dedicated to the production of viral DNA and of viralstructural proteins (Tooze, 1981).

The E1 region of adenovirus is the first region of adenovirus expressedafter infection of the target cell. This region consists of twotranscriptional units, the E1A and E1B genes, which both are requiredfor oncogenic transformation of primary (embryonal) rodent cultures. Themain functions of the E1A gene products are:

i) to induce quiescent cells to enter the cell cycle and resume cellularDNA synthesis, and

ii) to transcriptionally activate the E1B gene and the other earlyregions (E2, E3, E4). Transfection of primary cells with the E1A genealone can induce unlimited proliferation (immortalization), but does notresult in complete transformation. However, expression of E1A in mostcases results in induction of programmed cell death (apoptosis), andonly occasionally immortalization is obtained (Jochemsen et al., 1987).Co-expression of the E1B gene is required to prevent induction ofapoptosis and for complete morphological transformation to occur. Inestablished immortal cell lines, high level expression of E1A can causecomplete transformation in the absence of E1B (Roberts et al., 1985).

The E1B coded protein assist E1A in redirecting the cellular functionsto allow viral replication. The E1B 55 kD and E4 33 kD proteins, whichform a complex that is essentially localized in the nucleus, function ininhibiting the synthesis of host proteins and in facilitating theexpression of viral genes. Their main influence is to establishselective transport of viral mRNAs from the nucleus to the cytoplasm,concomittantly with the onset of the late phase of infection. The E1B 21kD protein is important for correct temporal control of the productiveinfection cycle, thereby preventing premature death of the host cellbefore the virus life cycle has been completed. Mutant viruses incapableof expressing the E1B 21 kD gene-product exhibit a shortened infectioncycle that is accompanied by excessive degradation of host cellchromosomal DNA (deg-phenotype) and in an enhanced cytopathic effect(cyt-phenotype) (Telling et al., 1994). The deg and cyt phenotypes aresuppressed when in addition the E1A gene is mutated, indicating thatthese phenotypes are a function of E1A (white et al., 1988).Furthermore, the E1B 21 kDa protein slows down the rate by which E1Aswitches on the other viral genes. It is not yet known through whichmechanisms) E1B 21 kD quenches these E1A dependent functions.

Vectors derived from human adenoviruses, in which at least the E1 regionhas been deleted and replaced by a gene of interest, have been usedextensively for gene therapy experiments in the pre-clinical andclinical phase.

As stated before all adenovirus vectors currently used in gene therapyhave a deletion in the E1 region, where novel genetic information can beintroduced. The E1 deletion renders the recombinant virus replicationdefective (Stratford-Perricaudet and Perricaudet, 1991). We havedemonstrated that recombinant adenoviruses are able to efficientlytransfer recombinant genes to the rat liver and airway epithelium ofrhesus monkeys (Bout et al., 1994b; Bout et al., 1994a). In addition, we(Vincent et al., 1996a; Vincent et al., 1996b) and others (see e.g.Haddada et al., 1993) have observed a very efficient in vivo adenovirusmediated gene transfer to a variety of tumor cells in vitro and to solidtumors in animals models (lung tumors, glioma) and human xenografts inimmunodeficient mice (lung) in vivo (reviewed by Blaese et al., 1995).

In contrast to for instance retroviruses, adenoviruses a) do notintegrate into the host cell genome; b) are able to infect non-dividingcells and c) are able to efficiently transfer recombinant genes in vivo(Brody and Crystal, 1994). Those features make adenoviruses attractivecandidates for in vivo gene transfer of, for instance, suicide orcytokine genes into tumor cells.

However, a problem associated with current recombinant adenovirustechnology is the possibility of unwanted generation of replicationcompetent adenovirus (RCA) during the production of recombinantadenovirus (Lochmuller et al., 1994; Imler et al., 1996). This is causedby homologous recombination between overlapping sequences from therecombinant vector and the adenovirus constructs present in thecomplementing cell line, such as the 293 cells (Graham et al., 1977).RCA in batches to be used in clinical trials is unwanted because RCA i)will replicate in an uncontrolled fashion; ii) can complementreplication defective recombinant adenovirus, causing uncontrolledmultiplication of the recombinant adenovirus and iii) batches containingRCA induce significant tissue damage and hence strong pathological sideeffects (Lockmuller et al., 1994). Therefore, batches to be used inclinical trials should be proven free of RCA (Ostrove, 1994). In oneaspect of the invention this problem in virus production is solved inthat we have developed packaging cells that have no overlappingsequences with a new basic vector and thus are suited for safe largescale production of recombinant adenoviruses one of the additionalproblems associated with the use of recombinant adenovirus vectors isthe host-defence reaction against treatment with adenovirus.

Briefly, recombinant adenoviruses are deleted for the E1 region (seeabove). The adenovirus E1 products trigger the transcription of theother early genes (E2, E3, E4), which consequently activate expressionof the late virus genes. Therefore, it was generally thought that E1deleted vectors would not express any other adenovirus genes. However,recently it has been demonstrated that some cell types are able toexpress adenovirus genes in the absence of E1 sequences. This indicates,that some cell types possess the machinery to drive transcription ofadenovirus genes. In particular, it was demonstrated that such cellssynthesize E2A and late adenovirus proteins.

In a gene therapy setting, this means that transfer of the therapeuticrecombinant gene to somatic cells not only results in expression of thetherapeutic protein but may also result in the synthesis of viralproteins. Cells that express adenoviral proteins are recognized andkilled by Cytotoxic T Lymphocytes, which thus a) eradicates thetransduced cells and b) causes inflammations (Bout et al., 1994a;Engelhardt et al., 1993; Simon et al, 1993). As this adverse reaction ishampering gene therapy, several solutions to this problem have beensuggested, such as a) using immunosuppressive agents after treatment; b)retainment of the adenovirus E3 region in the recombinant vector (seepatent application EP 95202213) and c) and using ts mutants of humanadenovirus, which have a point mutation in the E2A region (patentWO/28938).

However, these strategies to circumvent the immune response have theirlimitations.

The use of ts mutant recombinant adenovirus diminishes the immuneresponse to some extent, but was less effective in preventingpathological responses in the lungs (Engelhardt et al., 1994a).

The E2A protein may induce an immune response by itself and it plays apivotal role in the switch to the synthesis of late adenovirus proteins.Therefore, it is attractive to make recombinant adenoviruses which aremutated in the E2 region, rendering it temperature sensitive (ts), ashas been claimed in patent application WO/28938.

A major drawback of this system is the fact that, although the E2protein is unstable at the non-permissive temperature, the immunogenicprotein is still being synthesized. In addition, it is to be expectedthat the unstable protein does activate late gene expression, albeit toa low extent. ts125 mutant recombinant adenoviruses have been tested,and prolonged recombinant gene expression was reported (Yang et al.,1994b; Engelhardt et al., 1994a; Engelhardt et al., 1994b; Yang et al.,1995). However, pathology in the lungs of cotton rats was still high(Engelhardt et al., 1994a), indicating that the use of ts mutantsresults in only a partial improvement in recombinant adenovirustechnology. Others (Fang et al., 1996) did not observe prolonged geneexpression in mice and dogs using ts125 recombinant adenovirus. Anadditional difficulty associated with the use of ts125 mutantadenoviruses is that a high frequency of reversion is observed. Theserevertant are either real revertants or the result of second sitemutations (Kruijer et al., 1983; Nicolas et al., 1981). Both types ofrevertants have an E2A protein that functions at normal temperature andhave therefore similar toxicity as the wild-type virus.

In another aspect of the present invention we therefore delete E2Acoding sequences from the recombinant adenovirus genome and transfectthese E2A sequences into the (packaging) cell lines containing E1sequences to complement recombinant adenovirus vectors.

Major hurdles in this approach are a) that E2A should be expressed tovery high levels and b) that E2A protein is very toxic to cells.

The current invention in yet another aspect therefore discloses uses ofthe ts125 mutant E2A gene, which produces a protein that is not able tobind DNA sequences at the non permissive temperature. High levels ofthis protein may be maintained in the cells (because it is not toxic atthis temperature) until the switch to the permissive temperature ismade. This can be combined with placing the mutant E2A gene under thedirection of an inducible promoter, such as for instance tet,methallothionein, steroid inducible promoter, retinoic acid β-receptoror other inducible systems. However in yet another aspect of theinvention, the use of an inducible promoter to control the moment ofproduct of toxic wild-type E2A is disclosed.

Two salient additional advantages of E2A-deleted recombinant adenovirusare the increased capacity to harbor heterologous sequences and thepermanent selection for cells that express the mutant E2A. This secondadvantage relates to the high frequency of reversion of ts125 mutation:when reversion occurs in a cell line harboring ts125 E2A, this will belethal to the cell. Therefore, there is a permanent selection for thosecells that express the ts125 mutant E2A protein. In addition, as we inone aspect of the invention generate E2A-deleted recombinant adenovirus,we will not have the problem of reversion in our adenoviruses.

In yet another aspect of the invention as a further improvement the useof non-human cell lines as packaging cell lines is disclosed.

For GMP production of clinical batches of recombinant viruses it isdesirable to use a cell line that has been used widely for production ofother biotechnology products. Most of the latter cell lines are frommonkey origin, which have been used to produce e.g. vaccines. Thesecells can not be used directly for the production of recombinant humanadenovirus, as human adenovirus can not or only to low levels replicatein cells of monkey origin. A block in the switch of early to late phaseof adenovirus lytic cycle is underlying defective replication. However,host range mutations in the human adenovirus genome are described(hr400-404) which allow replication of human viruses in monkey cells.These mutations reside in the gene encoding E2A protein (Klessig andGrodzicker, 1979; Klessig et al., 1984; Rice and Klessig, 1985) (Klessiget al., 1984). Moreover, mutant viruses have been described that harborboth the hr and temperature-sensitive ts125 phenotype (Brough et al.,1985; Rice and Klessig, 1985).

We therefore generate packaging cell lines of monkey origin (e.g. VERO,CV1) that harbor:

a. E1 sequences, to allow replication of E1/E2 defective adenoviruses,and

b. E2A sequences, containing the hr mutation and the ts 125 mutation,named ts400 (Brough et al., 1985; Rice and Klessig, 1985) to preventcell death by E2A overexpression, and/or

c. E2A sequences, must containing the hr mutation, under the control ofan inducible promoter, and/or

d. E2A sequences, containing the hr mutation and the ts 125 mutation(ts400), under the control of an inducible promoter

Furthermore we disclose the construction of novel and improvedcombinations of (novel and improved) packaging cell lines and (novel andimproved) recombinant adenovirus vectors. We provide:

1. a novel packaging cell line derived from diploid human embryonicretinoblasts (HER) that harbors nt. 80-5788 of the Ad5 genome. This cellline, named 911; deposited under no 95062101 at the ECACC, has manycharacteristics that make it superior to the commonly used 293 cells(Fallaux et al., 1996).

2. novel packaging cell lines that express just E1A genes and not E1Bgenes. Established cell lines (and not human diploid cells of which 293and 911 cells are derived) are able to express E1A to high levelswithout undergoing apoptotic cell death, as occurs in human diploidcells that express E1A in the absence of E1B. Such cell lines are ableto trans-complement E1B-defective recombinant adenoviruses, becauseviruses mutated for E1B 21 kD protein are able to complete viralreplication even faster than wild-type adenoviruses (Telling et al.,1994). The constructs are described in detail below, and graphicallyrepresented in FIGS. 1-5. The constructs are transfected into thedifferent established cell lines and are selected for high expression ofE1A. This is done by operatively linking a selectable marker gene (e.g.NEO gene) directly to the E1B promoter. The E1B promoter istranscriptionally activated by the E1A gene product and thereforeresistance to the selective agent (e.g. G418 in the case NEO is used asthe selection marker) results in direct selection for desired expressionof the E1A gene

3. Packaging constructs that are mutated or deleted for E1B 21 kD, butjust express the 55 kD protein.

4. Packaging constructs to be used for generation of complementingpackaging cell lines from diploid cells (not exclusively of humanorigin) without the need of selection with marker genes. These cells areimmortalized by expression of E1A. However, in this particular caseexpression of E1B is essential to prevent apoptosis induced by E1Aproteins. Selection of E1 expressing cells is achieved by selection forfocus formation (immortalization), as described for 293 cells (Graham etal., 1977) and 911 calls (Fallaux et al, 1996), that are E1-transformedhuman embryonic kidney (HEK) cells and human embryonic retinoblasts(HER), respectively.

5. After transfection of HER cells with construct pIG.E1B (FIG. 4),seven independent cell lines could be established. These cell lines weredesignated PER.C1, PER.C3, PER.C4, PER.C5, PER.C6, PER.C8 and PER.C9.PER denotes PGK-E1-Retinoblasts. These cell lines express E1A and E1Bproteins, are stable (e.g. PER.C6 for more than 57 passages) andcomplement E1 defective adenovirus vectors. Yields of recombinantadenovirus obtained on PER cells are a little higher than obtained on293 cells. One of these cell lines (PER.C6) has been deposited at theECACC under number 96022940.

6. New adenovirus vectors with extended E1 deletions (deletionnt.459-3510). Those viral vectors lack sequences homologous to E1sequences in said packaging cell lines. These adenoviral vectors containpIX promoter sequences and the pIX gene, as pIX (from its naturalpromoter sequences) can only b expressed from the vector and not bypackaging cells (Matsui et al, 1986, Hoeben and Fallaux, pers.comm.;Imler et al., 1996).

7. E2A expressing packaging cell lines preferably based on either E1Aexpressing established cell lines or E1A-E1B expressing diploid cells(see under 2-4). E2A expression is either under the control of aninducible promoter or the E2A ts125 mutant is driven by either aninducible or a constitutive promoter.

8. Recombinant adenovirus vectors as described before (see 6) butcarrying an additional deletion of E2A sequences.

9. Adenovirus packaging cells from monkey origin that are able totrans-complement E1-defective recombinant adenoviruses. They arepreferably co-transfected with pIG.E1AE1B and pIG.NEO, and selected forNEO resistance. Such cells expressing E1A and E1B are able totranscomplement E1 defective recombinant human adenoviruses, but will doso inefficiently because of a block of the synthesis of late adenovirusproteins in cells of monkey origin (Klessig and Grodzicker, 1979). Toovercome this problem, we generate recombinant adenoviruses that harbora host-range mutation in the E2A gene, allowing human adenoviruses toreplicate in monkey cells. Such viruses are generated as described inFIG. 12, except DNA from a hr-mutant is used for homologousrecombination.

10. Adenovirus packaging cells from monkey origin as described under 9,except that they will also be co-transfected with E2A sequencesharboring the hr-mutation. This allows replication of human adenoviruseslacking E1 and E2A (see under 8). E2A in these cell lines is eitherunder the control of an inducible promoter or the tsE2A mutant is used.In the latter case, the E2A gene will thus carry both the ts mutationand the hr mutation (derived from ts400). Replication competent humanadenoviruses have been described that harbor both mutations (Brough etal., 1985; Rice and Klessig, 1985).

A further aspect of the invention provides otherwise improved adenovirusvectors, as well as novel strategies for generation and application ofsuch vectors and a method for the intracellular amplification of linearDNA fragments in mammalian cells.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures and drawings may help to understand the invention:

FIG. 1 illustrates the construction of pBS.PGK.PCRI;

FIG. 2 illustrates the construction of pIG.E1A.E1B.X;

FIG. 3 illustrates the construction of pIG.E1A.NEO;

FIG. 4 illustrates the construction of pIG.E1A.E1B;

FIG. 5 illustrates the construction of pIG.NEO;

FIG. 6 illustrates the transformation of primary baby rate kidney (BRK)cells by adenovirus packaging constructs;

FIG. 7 illustrates a Western blot analysis of A549 clones transfectedwith pIG.E1A.NEO and human embryonic retinoblasts (HER cells)transfected with pIG.E1A.E1B (PER clones);

FIG. 8 illustrates a Southern blot analysis of 293, 911 and PER celllines. Cellular DNA was extracted, Hind III digested, electrophoresedand transferred to Hybond N+membranes (Amersham);

FIG. 9 illustrates the transfection efficiency of PER.C3, PER.C5,PER.C6™ and 911 cells;

FIG. 10 illustrates construction of adenovirus vector, pMLPI.TK.pMLPI.TK designed to have no sequence overlap with the packagingconstruct pIG.E1A.E1B;

FIGS. 11a and 11b illustrate new adenovirus packaging constructs do nothave sequence overlap with new adenovirus vectors;

FIG. 12 illustrate the generation of recombinant adenovirus,IG.Ad.MLPI.TK;

FIG. 13 illustrates the adenovirus double-stranded DNA genome indicatingthe approximate locations of E1, E2, E3, E4, and L regions;

FIG. 14 illustrates the adenovirus genome is shown in the top left withthe origins or replication located within the left and right ITRs at thegenome ends;

FIG. 15 illustrates a potential hairpin conformation of asingle-stranded DNA molecule that contains the HP/asp sequence;

FIG. 16 illustrates a diagram of pICLhac;

FIG. 17 illustrates a diagram of pICLhaw;

FIG. 18 illustrates a schematic representation of pICLI;

FIG. 19 is a diagram of pICL; and

FIG. 20 recites the nucleotide sequence of pICL 5620BPS DNA (circular).

DETAILED DESCRIPTION OF THE INVENTION

The so-called "minimal" adenovirus vectors according to the presentinvention retain at least a portion of the viral genome that is requiredfor encapsidation of the genome into virus particles (the encapsidationsignal), as well as at least one copy of at least a functional part or aderivative of the Inverted Terminal Repeat (ITR), that is DNA sequencesderived from the termini of the linear adenovirus genome. The vectorsaccording to the present invention will also contain a transgene linkedto a promoter sequence to govern expression of the transgene. Packagingof the so-called minimal adenovirus vector can be achieved byco-infection with a helper virus or, alternatively, with a packagingdeficient replicating helper system as described below.

Adenovirus-derived DNA fragments that can replicate in suitable celllines and that may serve as a packaging deficient replicating helpersystem are generated as follows. These DNA fragments retain at least aportion of the transcribed region of the "late" transcription unit ofthe adenovirus genome and carry deletions in at least a portion of theE1 region and deletions in at least a portion of the encapsidationsignal. In addition, these DNA fragments contain at least one copy of aninverted terminal repeat (ITR). At one terminus of the transfected DNAmolecule an ITR is located. The other end may contain an ITR, oralternatively, a DNA sequence that is complementary to a portion of thesame strand of the DNA molecule other than the ITR. If, in the lattercase, the two complementary sequences anneal, the free 3'-hydroxyl groupof the 3' terminal nucleotide of the hairpin-structure can serve as aprimer for DNA synthesis by cellular and/or adenovirus-encoded DNApolymerases, resulting in conversion into a double-stranded form of atleast a portion of the DNA molecule. Further replication initiating atthe ITR will result in a linear double-stranded DNA molecule, that isflanked by two ITR's, and is larger than the original transfected DNAmolecule (see FIG. 13). This molecule can replicate itself in thetransfected cell by virtue of the adenovirus proteins encoded by the DNAmolecule and the adenovirus proteins encoded by the DNA molecule and theadenoviral and cellular proteins encoded by genes in the host-cellgenome. This DNA molecule can not be encapsidated due to its large size(greater than 39000 base pairs) or due to the absence of a functionalencapsidation signal. This DNA molecule is intended to serve as a helperfor the production of defective adenovirus vectors in suitable celllines.

The invention also comprises a method for the amplification of linearDNA fragments of variable size in suitable mammalian cells. These DNAfragments contain at least one copy of the ITR at one of the termini ofthe fragment. The other end may contain an ITR, or alternatively, a DNAsequence that is complementary to a portion of the same strand of theDNA molecule other than the ITR. If, in the latter case, the twocomplementary sequences anneal, the free 3'-hydroxyl group of the 3'terminal nucleotide of the hairpin-structure can serve as a primer forDNA synthesis by cellular and/or adenovirus-encoded DNA polymerases,resulting in conversion of the displaced stand into a double strandedform of at least a portion of the DNA molecule. Further replicationinitiating at the ITR will result in a linear double-stranded DNAmolecule, that is flanked by two ITR's, which is larger than theoriginal transfected DNA molecule. A DNA molecule that contains ITRsequences at both ends can replicate itself in transfected cells byvirtue of the presence of at least the adenovirus E2 proteins (viz. theDNA-binding protein (DBP), the adenovirus DNA polymerase (Ad-pol), andthe preterminal protein (pTP). The required proteins may be expressedfrom adenovirus genes on the DNA molecule itself, from adenovirus E2genes integrated in the host-cell genome, or from a replicating helperfragment as described above.

Several groups have shown that the presence of ITR sequences at the endof DNA molecules are sufficient to generate adenovirus minichromosomesthat can replicate, if the adenovirus-proteins required for replicationare provided in trans e.g. by infection with a helpervirus (Hu et al.,1992); (Wang and Pearson, 1985); (Hay et al., 1984). Hu et al., (1992)observed the presence and replication of symmetrical adenovirusminichromosome-dimers after transfection of plasmids containing a singleITR. The authors were able to demonstrate that these dimericminichromosomes arize after tail-to-tail ligation of the single ITR DNAmolecules. In DNA extracted from defective adenovirus type 2 particles,dimeric molecules of various sizes have also been observed usingelectron-microscopy (Daniell, 1976). It was suggested that theincomplete genomes were formed by illegitimate recombination betweendifferent molecules and that variations in the position of the sequenceat which the illegitimate base pairing occurred were resonsible for theheterogeneous nature of the incomplete genomes. Based on this mechanismit was speculated that, in theory, defective molecules with a totallength of up to two times the normal genome could be generated. Suchmolecules could contain duplicated sequences from either end of thegenome. However, no DNA molecules larger than the full-length virus werefound packaged in the defective particles (Daniell, 1976). This can beexplained by the size-limitations that apply to the packaging. Inaddition, it was observed that in the virus particles DNA-molecules witha duplicated left-end predominated over those containing the right-endterminal (Daniell, 1976). This is fully explained by the presence of theencapsidation signal near that left-end of the genome (Grable andHearing, 1990; Grable and Hearing, 1992; Hearing et al., 1987).

The major problems associated with the current adenovirus-derivedvectors are:

A) The strong immunogenicity of the virus particle

B) The expression of adenovirus genes that reside in the adenoviralvectors, resulting in a Cytotoxic T-cell response against the transducedcells.

C) The low amount of heterologous sequences that can be accommodated inthe current vectors (Up to maximally approx. 8000 bp. of heterologousDNA).

Ad A) The strong immunogenicity of the adenovirus particle results in animmunological response of the host, even after a single administrationof the adenoviral vector. As a result of the development of neutralizingantibodies, a subsequent administration of the virus will be lesseffective or even completely ineffective. However, a prolonged orpersistent expression of the transferred genes will reduce the number ofadministrations required and may bypass the problem.

Ad B) Experiments performed by Wilson and collaborators havedemonstrated that after adenovirus-mediated gene transfer intoimmunocompetent animals, the expression of the transgene graduallydecreases and disappears approximately 2-4 weeks post-infection (Yang etal., 1994a; Yang et al., 1994b). This is caused by the development of aCytotoxic T-Cell (CTL) response against the transduced cells. The CTLswere directed against adenovirus proteins expressed by the viralvectors. In the transduced cells synthesis of the adenovirus DNA-bindingprotein (the E2A-gene product), penton and fiber proteins (late-geneproducts) could be established. These adenovirus proteins, encoded bythe viral vector, were expressed despite deletion of the E1 region. Thisdemonstrates that deletion of the E1 region is not sufficient tocompletely prevent expression of the viral genes (Engelhardt et al.,1994a).

Ad C) Studies by Graham and collaborators have demonstrated thatadenoviruses are capable of encapsidating DNA of up to 105% of thenormal genome size (Bett et al., 1993). Larger genomes tend to beinstable resulting in loss of DNA sequences during propagation of thevirus. Combining deletions in the E1 and E3 regions of the viral genomesincreases the maximum size of the foreign that can be encapsidated toapprox. 8.3 kb. In addition, some sequences of the E4 region appear tobe dispensable for virus growth (adding another 1.8 kb to the maximumencapsidation capacity). Also the E2A region can be deleted from thevector, when the E2A gene product is provided in trans in theencapsidation cell line, adding another 1.6 kb. It is, however, unlikelythat the maximum capacity of foreign DNA can be significantly increasedfurther than 12 kb.

We developed a new strategy for the generation and production ofhelperfree-stocks of recombinant adenovirus vectors that can accommodateup to 38 kb of foreign DNA. Only two functional ITR sequences, andsequences than can function as an encapsidation signal need to be partof the vector genome. Such vectors are called minimal adenovectors. Thehelper functions for the minimal adenovectors are provided in trans byencapsidation defective-replication competent DNA molecules that containall the viral genes encoding the required gene products, with theexception of those genes that are present in the host-cell genome, orgenes that reside in the vector genome.

The applications of the disclosed inventions are outlined below and willbe illustrated in the experimental part, which is only intended for saidpurpose, and should not be used to reduce the scope of the presentinvention as understood by the person skilled in the art.

Use of the IG packaging constructs Diploid cells.

The constructs, in particular pIG.E1A.E1B, will be used to transfectdiploid human cells, such as Human Embryonic Retinoblasts (HER), HumanEmbryonic Kidney cells (HEK), and Human Embryonic Lung cells (HEL).Transfected cells will be selected for transformed phenotype (focusformation) and tested for their ability to support propagation ofE1-deleted recombinant adenovirus, such as IG.Ad.MLPI.TK. Such celllines will be used for the generation and (large-scale) production ofE1-delete recombinant adenoviruses. Such cells, infected withrecombinant adenovirus are also intended to be used in vivo as a localproducer of recombinant adenovirus, e.g. for the treatment of solidtumors.

911 cells are used for the titration, generation and production ofrecombinant adenovirus vectors (Fallaux et al., 1996).

HER cells transfected with pIG.E1A.E1B has resulted in 7 independentclones (called PER cells). These clones are used for the production ofE1 deleted (including non-overlapping adenovirus vectors) or E1defective recombinant adenovirus vectors and provide the basis forintroduction of e.g. E2B or E2A constructs (e.g. ts125E2A, see below),E4 etc., that will allow propagation of adenovirus vectors that havemutations in e.g. E2A or E4.

In addition, diploid cells of other species that are permissive forhuman adenovirus, such as the cotton rat (Sigmodon hispidus) (Pacini etal., 1984), Syrian hamster (Morin et al., 1987) or chimpanzee (Levreroet al., 1991), will be immortalized with these constructs. Such cells,infected with recombinant adenovirus, are also intended to be used invivo for the local production of recombinant adenovirus, e.g. for thetreatment of solid tumors.

Established cells.

The constructs, in particular pIG.E1A.NEO, can be used to transfectestablished cells, e.g. A549 (human bronchial carcinoma), KB (oralcarcinoma), MRC-5 (human diploid lung cell line) or GLC cell lines(small cell lung cancer) (de Leij et al., 1985; Postmus et al., 1988)and selected for NEO resistance. Individual colonies of resistant cellsare isolated and tested for their capacity to support propagation ofE1-deleted recombinant adenovirus, such as IG.Ad.MLPI.TK. Whenpropagation of E1 deleted viruses on E1A containing cells is possible,such cells can be used for the generation and production of E1-deletedrecombinant adenovirus. They are also be used for the propagation of E1Adeleted/E1B retained recombinant adenovirus.

Established cells can also be co-transfected with pIG.E1A.E1B andpIG.NEO (or another NEO containing expression vector). Clones resistantto G418 are tested for their ability to support propagation of E1deleted recombinant adenovirus, such as IG.Ad.MLPI.TK and used for thegeneration and production of E1 deleted recombinant adenovirus and willbe applied in vivo for local production of recombinant virus, asdescribed for the diploid cells (see above).

All cell lines, including transformed diploid cell lines orNEO-resistant established lines, can be used as the basis for thegeneration of `next generation` packaging cells lines, that supportpropagation of E1-defective recombinant adenoviruses, that also carrydeletions in other genes, such as E2A and E4. Moreover, they willprovide the basis for the generation of minimal adenovirus vectors asdisclosed herein.

E2 expressing cell lines

Packaging cells expressing E2A sequences are and will be used for thegeneration and (large scale) production of E2A-deleted recombinantadenovirus.

The newly generated human adenovirus packaging cell lines or cell linesderived from species permissive for human adenovirus (E2A or ts125E2A;E1A+E2A; E1A+E1B-E2A; E1A-E2A/ts125; E1A+E1B-E2A/ts125) ornon-permissive cell lines such as monkey cells (hrE2A or hr-ts125E2A;E1A+hrE2A; E1A+E1B-hrE2A; E1A+hrE2A/ts125; E1A-E1B+hrE2A/ts125) are andwill be used for the generation and (large scale) production of E2Adeleted recombinant adenovirus vectors. In addition, they will beapplied in vivo for local production of recombinant virus, as describedfor the diploid cells (see above).

Novel adenovirus vectors.

The newly developed adenovirus vectors harboring an E1 deletion of nt.459-3510 will be used for gene transfer purposes. These vectors are alsothe basis for the development of further deleted adenovirus vectors thatare mutated for e.g. E2A, E2B or E4. Such vectors will be generated e.g.on the newly developed packaging cell lines described above (see 1-3).

Minimal adenovirus packaging system

We disclose adenovirus packaging constructs (to be used for thepackaging of minimal adenovirus vectors may have the followingcharacteristics:

a. the packaging construct replicates

b. the packaging construct can not be packaged because the packagingsignal is deleted

c. the packaging construct contains an internal hairpin-forming sequence(see section `Experimental; suggested hairpin` see FIG. 15)

d. because of the internal hairpin structure, the packaging construct isduplicated, that is the DNA of the packaging construct becomes twice aslong as it was before transfection into the packaging cell (in oursample it duplicates from 35 kb to 70 kb). This duplication alsoprevents packaging. Note that this duplicated DNA molecule has ITR's atboth termini (see e.g. FIG. 13)

e. this duplicated packaging molecule is able to replicate like a`normal adenovirus` DNA molecule

f. the duplication of the genome is a prerequisite for the production ofsufficient levels of adenovirus proteins, required to package theminimal adenovirus vector

g. the packaging construct has no overlapping sequences with the minimalvector or cellular sequences that may lead to generation of RCA byhomologous recombination.

This packaging system will be used to produce minimal adenovirusvectors. The advantages of minimal adenovirus vectors e.g. for genetherapy of vaccination purposes, are well known (accommodation of up to38 kb; gutting of all potentially toxic and immunogenic adenovirusgenes).

Adenovirus vectors containing mutations in essential genes (includingminimal adenovirus vectors) can also be propagated using this system.

Use of intracellular E2 expressing vectors.

Minimal adenovirus vectors are generated using the helper functionsprovided in trans by packaging-deficient replicating helper molecules.The adenovirus-derived ITR sequences serve as origins of DNA replicationin the presence of at least the E2-gene products. When the E2 geneproducts are expressed from genes in the vector genome (N.B. the gene(s)must be driven by an E1-independent promoter), the vector genome canreplicate in the target cells. This will allow an significantlyincreased number of template molecules in the target cells, and, as aresult an increased expression of the genes of interest encoded by thevector. This is of particular interest for approaches of gene therapy incancer.

Applications of intracellular amplification of linear DNA fragments.

A similar approach could also be taken if amplification of linear DNAfragments is desired. DNA fragments of known or unknown sequence couldbe amplified in cells containing the E2-gene products if at least oneITR sequence is located near or at its terminus. There are no apparentconstrains on the size of the fragment. Even fragments much larger thanthe adenovirus genome (36 kb) should be amplified using this approach.It is thus possible to clone large fragments in mammalian cells withouteither shuttling the fragment into bacteria (such as E. coli) or use thepolymerase chain reaction (P.C.R.). At the end stage of an productiveadenovirus infection a single cell can contain over 100,000 copies ofthe viral genome. In the optimal situation, the linear DNA fragments canbe amplified to similar levels. Thus, one should be able to extract morethan 5 μg of DNA fragment per 10 million cells (for a 35-kbp fragment).This system can be used to express heterologous proteins equivalent tothe Simian Virus 40-based COS-cell system) for research or fortherapeutic purposes. In addition, the system can be used to identifygenes in large fragments of DNA. Random DNA fragments may be amplified(after addition of ITRs) and expressed during intracellularamplification. Election or, selection of those cells with the desiredphenotype can be used to enrich the fragment of interest and to isolatethe gene.

EXPERIMENTAL

Generation of cell lines able to transcomplement E1 defectiverecombinant adenovirus vectors.

1. 911 cell line

We have generated a cell line that harbors E1 sequences of adenovirustype 5, able to trans-complement E1 deleted recombinant adenovirus(Fallaux et al., 1996).

This cell line was obtained by transfection of human diploid humanembryonic retinoblasts (HER) with pAd5XhoIC, that contains nt. 80-5788of Ad5; one of the resulting transformants was designated 911. This cellline has been shown to be very useful in the propagation of E1 defectiverecombinant adenovirus. It was found to be superior to the 293 cells.Unlike 293 cells, 911 cells lack a fully transformed phenotype, whichmost likely is the cause of performing better as adenovirus packagingline:

plaque assays can be performed faster (4-5 days instead of 8-14 days on293)

monolayers of 911 cells survive better under agar overlay as requiredfor plaque assays

higher amplification of E1-deleted vectors

In addition, unlike 293 cells that were transfected with shearedadenoviral DNA, 911 cells were transfected using a defined construct.Transfection efficiencies of 911 cells are comparable to those of 293.

New packaging constructs.

Source of adenovirus sequences.

Adenovirus sequences are derived either from pAd5.SalB, containing nt.80-9460 of human adenovirus type 5 (Bernards et al., 1983) or fromwild-type Ad5 DNA.

pAd5.SalB was digested with SalI and XhoI and the large fragment wasreligated and this new clone was named pAd5.X/S.

The pTN construct (constructed by Dr. R. Vogels, IntroGene, TheNetherlands) was used as a source for the human PGK promoter and the NEOgene.

Human PGK promoter and NEO^(R) gene.

Transcription of E1A sequences in the new packaging constructs is drivenby the human PGK promoter (Michelson et al., 1983; Singer-Sam et al.,1984), derived from plasmid pTN (gift of R. Vogels), which uses pUC119(Vieira and Messing, 1987) as a backbone. This plasmid was also used asa source for NEO gene fused to the Hepatitis B Virus (HBV)poly-adenylation signal.

Fusion of PGK promoter to E1 genes (FIG. 1)

In order to replace the E1 sequences of Ad5 (ITR, origin of replicationand packaging signal) by heterologous sequences we have amplified E1sequences (nt. 459 to nt. 960) of Ad5 by PCR, using primers Ea-1 (SEQ IDNO:1) and Ea-2 (SEQ ID NO:2) (see Table I). The resulting PCR productwas digested with ClaI and ligated into Bluescript (Stratagene),predigested with ClaI and EcoRV, resulting in construct pBS.PCRI.

Vector pTN was digested with restriction enzymes EcoRI (partially) andScaI, and the DNA fragment containing the PGK promoter sequences wasligated into PBS.PCRI digested with ScaI and EcoRI. The resultingconstruct PBS.PGK.PCRI contains the human PGK promoter operativelylinked to Ad5 E1 sequences from nt.459 to nt. 916.

Construction of pIG.E1A.E1B.X (FIG. 2)

pIG.E1A.E1B.X was made by replacing the ScaI-BspEI fragment of pAT-X/Sby the corresponding fragment from PBS.PGK.PCRI (containing the PGKpromoter linked to E1A sequences).

pIG.E1A.E1B.X contains the E1A and E1B coding sequences under thedirection of the PGK promoter.

As Ad5 sequences from nt.459 to nt. 5788 are present in this construct,also pIX protein or adenovirus is encoded by this plasmid.

Construction of pIG.E1A.NEO (FIG. 3)

In order to introduce the complete E1B promoter and to fuse thispromoter in such a way that the AUG codon of E1B 21 kD exactly functionsas the AUG codon or NEO^(R), we amplified the E1B promoter using primersEa-3 (SEQ ID NO:3) Ep-2 (SEQ ID NO:5) where primer Ep-2 introduces anNcoI site in the PCR fragment. The resulting PCR fragment, named PCRII,was digested with HpaI and NcoI and ligated into pAT-X/S, which waspredigested with HpaI and with NcoI. The resulting plasmid wasdesignated pAT-X/S-PCR2. The NcoI-StuI fragment of pTN, containing theNEO gene and part of the Hepatitis B Virus (HBV) poly-adenylationsignal, was cloned into pAT-X/S-PCR2 (digested with NcoI and NruI). Theresulting construct: pAT-PCR2-NEO. The poly-adenylation signal wascompleted by replacing the ScaI-SalI fragment of pAT-PCR2-NEO by thecorresponding fragment of pTN (resulting in pAT-PCR2.NEO.p(A)). TheScaI-XbaI of pAT.PCR2.NEO.p (A) was replaced by the correspondingfragment of pIG.E1A.E1B-X, containing the PGK promoter linked to E1Agenes.

The resulting construct was named pIG.E1A.NEO, and thus contains Ad5 E1sequences nt.459 to nt. 1713) under the control of the human PGKpromoter.

Construction of pIG.EIA.E1B (FIG. 4)

pIG.E1A.E1B was made by amplifying the sequences encoding the N-terminalamino acids of E1B 55 kd using primers Eb-1 (SEQ ID NO:6) and Eb-2 (SEQID NO:7) (introduces a XhoI site). The resulting PCR fragment wasdigested with BglII and cloned into BglII/NruI of pAT-X/S, therebyobtaining pAT-PCR3.

pIG.E1A.E1B was constructed by introducing the HBV poly(A) sequences ofpIG.E1A.NEO downstream of E1B sequences of pAT-PCR3 by exchange ofXbaI-SalI fragment of pIG.E1A.NEO and the XbaI XhoI fragment ofpAT.PCR3.

pIG.E1A.E1B contains nt. 459 to nt. 3510 of Ad5, that encode the E1A andE1B proteins. The E1B sequences are terminated at the splice acceptor atnt.3511. No pIX sequences are present in this construct.

Construction of pIG.NEO (FIG. 5)

pIG.NEO was generated by cloning the HpaI-ScaI fragment of pIG.E1A.NEO,containing the NEO gene under the control of the Ad.5 E1B promoter, intopBS digested with EcoRV and ScaI.

This construct is of use when established cells are transfected withE1A.E1B constructs and NEO selection is required. Because NEO expressionis directed by the E1B promoter, NEO resistant cells are expected toco-express E1A, which also is advantageous for maintaining high levelsof expression of E1A during long-term culture of the cells.

Testing of constructs.

The integrity of the constructs pIG.E1A.NEO, pIG.E1A.E1B.X andpIG.E1A.E1B was assessed by restriction enzyme mapping; furthermore,parts of the constructs that were obtained by PCR analysis wereconfirmed by sequence analysis. No changes in the nucleotide sequencewere found.

The constructs were transfected into primary BRK (Baby Rat Kidney) cellsand tested for their ability to immortalize (pIG.E1A.NEO) or fullytransform (pAd5.XhoIC,pIG.E1A.E1B.X and pIG.E1A.E1B) these cells.

Kidneys of 6-day old WAG-Rij rats were isolated, homogenized andtrypsinized. Subconfluent dishes (diameter 5 cm) of the BRK cellcultures were transfected with 1 or 5 μg of pIG.NEO, pIG.E1A.NEO,pIG.E1A.E1B, pIG.E1A.E1B.X, pAd5XhoIC, or with pIG.E1A.NEO together withPDC26 (Van der Elsen et al., 1983), carrying the Ad5.E1B gene undercontrol of the SV40 early promoter. Three weeks post-transfection, whenfoci were visible, the dishes were fixed, Giemsa stained and the focicounted.

An overview of the generated adenovirus packaging constructs, and theirability to transform BRK, is presented in FIG. 6. The results indicatethat the constructs pIG.E1A.E1B and pIG.E1A.E1B.X are able to transformBRK cells in a dose-dependent manner. The efficiency of transformationis similar for both constructs and is comparable to what was found withthe construct that was used to make 911 cells, namely pAd5.XhoIC.

As expected, pIG.E1A.NEO was hardly able to immortalize BRK. However,co-transfection of an E1B expression construct (PDC26) did result in asignificant increase of the number of transformants (18 versus 1),indicating that E1A encoded by pIG.E1A.NEO is functional.

We conclude therefore, that the newly generated packaging constructs aresuited for the generation of new adenovirus packaging lines.

Generation of cell lines with new packaging constructs Cell lines andcell culture

Human A549 bronchial carcinoma cells (Shapiro et al., 1978), humanembryonic retinoblasts (HER), Ad5-E1-transfor-med human embryonic kidney(HEK) cells (293; Graham et al., 1977) cells and Ad5-transformed HERcells (911; Fallaux et al, 1996)) and PER cells were grown in Dulbecco'sModified Eagle Medium (DMEM) supplemented with 10% Fetal Calf Serum(FCS) and antibiotics in a 5% C02 atmosphere at 37° C. Cell culturemedia, reagents and sera were purchased from Gibco Laboratories (GrandIsland, N.Y.). Culture plastics were purchased from Greiner (Nurtingen,Germany) and Corning (Corning, N.Y.).

Viruses and virus techniques

The construction of adenoviral vectors IG.Ad.MLP.nls.lacZ,IG.Ad.MLP.luc, IG.Ad.MLP.TK and IG.Ad.CMV.TK is described in detail inpatent application EP 95202213.

The recombinant adenoviral vector IG.Ad.MLP.nls.lacZ contains the E.coli lacZ gene, encoding β-galactosidase, under control of the Ad2 majorlate promoter (MLP).IG.Ad.MLP.luc contains the firefly luciferase genedriven by the Ad2 MLP. Adenoviral vectors. IG.Ad.MLP.TK and ID.Ad.CMV.TKcontain the Herpes Simplex Virus thymidine kinase (TK) gene under thecontrol of the Ad2 MLP and the Cytomegalovirus (CMV) enhancer/promoter,respectively.

Transfections

All transfections were performed by calcium-phosphate precipitation DNA(Graham and Van der Eb, 1973) with the GIBCO Calcium PhosphateTransfection System (GIBCO BRL Life Technologies Inc., Gaithersburg,USA), according to the manufacturers protocol.

Western blotting

Subconfluent cultures of exponentially growing 293,911 andAd5-E1-transformed A549 and PER cells were washed with PBS and scrapedin Fos-RIPA buffer (10 mM Tris (pH 7,5), 150 mM NaCl, 1% NP40, 01%sodium dodecyl sulphate (SDS), 1% NA-DOC, 0.5 mM phenyl methyl sulphonylfluoride (PMSF), 0.5 mM trypsin inhibitor, 50 mM NaF and 1 mM sodiumvanadate). After 10 min. at room temperature, lysates were cleared bycentrifugation. Protein concentrations were measured with the Bioradprotein assay kit, and 25 μg total cellular protein was loaded on a12.5% SDS-PAA gel. After electrophoresis, proteins were transferred tonitrocellulose (1 h at 300 mA). Prestained standards (Sigma, USA) wererun in parallel. Filters were blocked with 1% bovine serum albumin (BSA)in TBST (10 mM Tris, pH 8, 15 mM NaCl, and 0.05% Tween-20) for 1 hour.First antibodies were the mouse monoclonal anti-Ad5-E1B-55-kDA antibodyA1C6 (Zantema et al., unpublished), the rat monoclonalanti-Ad5-E1B-221-kDa antibody C1G11 (Zantema et al., 1985). The secondantibody was a horseradish peroxidase-labeled goat anti-mouse antibody(Promega). Signals were visualized by enhanced chemoluminescence(Amersham Corp, UK).

Southern blot analysis

High molecular weight DNA was isolated and 10 μg was digested tocompletion and fractionated on a 0.7% agarose gel. Southern blottransfer to Hybond N+ (Amersham, UK) was performed with a 0.4 M NAOH,0.6 M NaCl transfer solution (Church and Gilbert, 1984). Hybridizationwas performed with a 2463-nt SspI-HindIII fragment from pAd5.SalB(Bernards et al., 1983). This fragment consists of Ad5 bp. 342-2805. Thefragment was radiolabeled with α-³² P-dCTP with the use of randomhexanucleotide primers and Klenow DNA polymerase. The southern blotswere exposed to a Kodak XAR-5 film at -80° C. and to a Phospho-Imagerscreen which was analyzed by B&L systems Molecular Dynamics software.

A549

Ad5-E1-transformed A549 human bronchial carcinoma cell lines weregenerated by transfection with pIG-E1A.NEO and selection for G418resistance. Thirty-one G418 resistant clones were established.Co-transfection of pIG.E1A.E1B with pIG.NEO yielded seven G418 resistantcell lines.

PER

Ad5-E1-transformed human embryonic retina (HER) cells were generated bytransfection of primery HER cells with plasmid pIG.E1A.E1B. Transformedcell lines were established from well-separated foci. We were able toestablish seven clonal cell lines, which we called PER.C1, PER.C3,PER.C4, PER.C5, PER.C6, PER.C8 and PER.C9.

One of the PER clones, namely PER.C6, has been deposited at the ECACCunder number 96022940.

Expression of Ad5 E1A and E1B genes in transformed A549 and PER cells

Expression of the Ad5 E1A and the 55-kDa and 21 kDa E1B proteins in theestablished A549 and PER cells was studied by means of Western blotting,with the use of monoclonal antibodies (mAb). Mab M73 recognizes the E1Aproducts, whereas Mabls AIC6 and C1G11 are directed against the 55-kDaand 21 kDa E1B proteins, respectively.

The antibodies did not recognize proteins in extracts from the parentalA549 or the primary HER cells (data not shown). None of the A549 clonesthat were generated by co-transfection of pIG.NEO and pIG.E1A.E1Bexpressed detectable levels of E1A or E1B proteins (not shown). Some ofthe A549 clones that were generated by transfection with pIG.E1A.NEOexpressed the Ad5 E1A proteins (FIG. 7), but the levels were much lowerthan those detected in protein lysates from 293 cells. The steady stateE1A levels detected in protein extracts from PER cells were much higherthan those detected in extracts from A549-derived cells. All PER celllines expressed similar levels of E1A proteins (FIG. 7). The expressionof the E1B proteins, particularly in the case of E1B 55 kDa, was motevariable. Compared to 911 and 293, the majority of the PER clonesexpress high levels of E1B 55 kDa and 21 kDa. The steady state level ofE1B 21 kDa was the highest in PER.C3. None of the PER clones lostexpression of the Ad5 E1 genes upon serial passage of the cells (notshown). We found that the level of E1 expression in PER cells remainedstable for at least 100 population doublings. We decided to characterizethe PER clones in more detail.

Southern analysis of PER clones

To study the arrangement of the Ad5-E1 encoding sequences in the PERclones we performed Southern analyses. Cellular DNA was extracted fromall PER clones, and from 293 and 911 cells. The DNA was digested withHindIII, which cuts once in the Ad5 E1 region. Southern hybridization onHindIII-digested DNA, using a radiolabeled Ad5-E1-specific proberevealed the presence of several integrated copies of pIG.E1A.E1B in thegenome of the PER clones. FIG. 8 shows the distribution pattern of E1sequences in the high molecular weight DNA of the different PER celllines. The copies are concentrated in a single band, which suggests thatthey are integrated as tandem repeats. In the case of PER.C3, C5, C6 andC9 we found additional hybridizing bands of low molecular weight thatindicate the presence of truncated copies of pIG.E1A.E1B. The number ofcopies was determined with the use of a Phospho-Imager. We estimated thePER.C1, C3, C4, C5, C6, C8 and C9 contain 2, 88, 5,4, 5, 5 and 3 copiesof the Ad5 E1 coding region, respectively, and that 911 and 293 cellscontain 1 and 4 copies of the Ad5 E1 sequences, respectively.

Transfection efficiency

Recombinant adenovectors are generated by co-transfection of adaptorplasmids and the large ClaI fragment of Ad5 into 293 cells (gee patentapplication EP 95202213). The recombinant virus DNA is formed byhomologous recombination between the homologous viral sequences that arepresent in the plasmid and the adenovirus DNA. The efficacy of thismethod, as well as that of alternative strategies, is highly dependenton the transfectability of the helper cells. Therefore, we compared thetransfection efficiencies of some of the PER clones with 911 cells,using the E. coli β-galactosidase-encoding lacZ gene as a reporter (FIG.9).

Production of Recombinant Adenovirus

Yields of recombinant adenovirus obtained after inoculation of 293, 911,PER.C3, PER.C5 and PER.C6 with different adenovirus vectors arepresented in Table II.

The results indicate that the yields obtained on PER cells are at leastas high as those obtained on the existing cell lines.

In addition, the yields of the novel adenovirus vector IG.Ad.MLPI.TK aresimilar or higher than the yields obtained for the other viral vectorson all cell lines tested.

Generation of New Adenovirus Vectors (FIG. 10)

The used recombinant adenovirus vectors (see patent application on EP95202213) are deleted for E1 sequences from 459 to nt. 3328.

As construct pE1a.E1B contains Ad5 sequences 459 to nt. 3510 there is asequence overlap of 183 nt. between E1B sequences in the packagingconstruct pIG.E1A.E1B and recombinant adenoviruses, such as e.g.IG.Ad.MLP.TK. The overlapping sequences were deleted from the newadenovirus vectors. In addition, non-coding sequences derived from lacZ,that are present in the original constructs, were deleted as well. Thiswas achieved (see FIG. 10) by PCR amplification of the SV40 poly(A)sequences from pMLP.TK using primers SV40-1 (SEQ ID NO:8) (introduces aBamHI site) and SV40-2 Ad-5-1 (SEQ ID NO:10) (introduces a BglII site).In addition, Ad5 sequences present in this construct were amplified fromnt. 2496 (Ad5, introduces a BglII site) to nt. 2779 (Ad5-2) (SEQ IDNO:11). Both PCR fragments were digested with BglII and were ligated.The ligation product was PCR amplified using primers SV40-1 and Ad5-2.The PCR product obtained was cut with BamHI and AflII and was ligatedinto pMLP.TK predigested with the same enzymes. The resulting construct,named pMLPI.TK, contains a deletion in adenovirus E1 sequences from nt459 to nt. 3510.

Packaging System

The combination of the new packaging construct pIG.E1A.E1B and therecombinant adenovirus pMLPI.TK, which do not have any sequence overlap,are presented in FIG. 11. In this figure, also the original situation ispresented, where the sequence overlap is indicated.

The absence of overlapping sequences between pIG.E1A.E1B and pMLPI.TK(FIG. 11a) excludes the possibility of homologous recombination betweenpackaging construct and recombinant virus, and is therefore asignificant improvement for production of recombinant adenovirus ascompared to the original situation.

In FIG. 11b the situation is depicted for pIG.E1A.NEO and IG.Ad.MLPI.TK.pIG.E1A.NEO when transfected into established cells, is expected to besufficient to support propagation of E1-deleted recombinant adenovirus.This combination does not have any sequence overlap, preventinggeneration of RCA by homologous recombination. In addition, thisconvenient packaging system allows the propagation of recombinantadenoviruses that are deleted just for E1A sequences and not for E1Bsequences. Recombinant adenoviruses expressing E1B in the absence of E1Aare attractive, as the E1B protein, in particular E1B 19 kD, is able toprevent infected human cells from lysis by Tumor Necrosis Factor (TNF)(Gooding et al., 1991).

Generation of Recombinant Adenovirus Derived From pMLPI.TK

Recombinant adenovirus was generated by co-transfection of 293 cellswith SalI linearized pMLPI.TK DNA and ClaI linearized Ad5 wt DNA. Theprocedure is schematically represented in FIG. 12.

Outline of the Strategy to Generate Packaging Systems For MinimalAdenovirus Vector

Name convention of the plasmids used:

P plasmid

I ITR (Adenovirus Inverted Terminal Repeat)

C Cytomegalovirus (CMV) Enhancer/Promoter Combination

L Firefly Luciferase Coding Sequence hac.haw Potential hairpin that canbe formed after digestion with restriction endonuclease Asp718 in itscorrect and in the reverse orientation, respectively (FIG. 15) (SEQ IDNO:22).

Eg.pICLhaw is a plasmid that contains the adenovirus ITR followed by theCMV-driven luceriferase gene and the Asp718 hairpin in the reverse(non-functional) orientation.

1.1 Demonstration of the competence of a synthetic DNA sequence, that iscapable of forming a hairpin-structure, to serve as a primer for reversestrand synthesis for the generation of double-stranded DNA molecules incells that contain and express adenovirus genes. Plasmids pICLhac,pICLhaw, pICLI and pICL were generated using standard techniques. Theschematic representation of these plasmids is shown in FIGS. 16-19.

Plasmid pICL is derived from the following plasmids:

nt.1-457 pMLP10 (Levrero et al., 1991)

nt.458-1218 pCMVβ (Clontech, EMBL Bank No. U02451)

nt.1219-3016 pMLP.luc (IntroGene, unpublished)

nt. 3017-5620 pBLCAT5 (Stein and Whelan, 1989) The plasmid has beenconstructed as follows:

The tet gene of plasmid pMLP10 has been inactivated by detection of theBamHI-SalI fragment, to generate pMLP10ΔSB. Using primer set PCR/MLP1(SEQ ID NO:14) and pCR/MLP3 (SEQ ID NO:16) a 210 bp fragment containingthe Ad5-ITR, flanked by a synthetic SalI restriction site was amplifiedusing pMLP10 DNA as the template. The PCR product was digested with theenzymes EcoRI and SgrAI to generate a 196 bp. fragment. PlasmidpMLP10ΔSB was digested with EcoRI and SgrAI to remove the ITR. Thisfragment was replaced by the EcoRI-SgrAI-treated PCR fragment togenerate pMLP/SAL. Plasmid pCMV-Luc was digested with PvuII tocompletion and recirculated to remove the SV40-derived poly-adenylationsignal and Ad5 sequences with exception of the Ad5 left-terminus. In theresulting plasmid, pCMV-lucΔAd, the Ad5 ITR was replaced by theSal-site-flanked ITR from plasmid pMLP/SAL by exchanging the XmnI-SacIIfragments. The resulting plasmid, pCMV-lucΔAd/SAL, the Ad5 left terminusand the CMV-driven luciferase gene were isolated as an SalI-SmaIfragment and inserted in the SalI and HpaI digested plasmid pBLCATS, toform plasmid pICL. Plasmid pICL is represented in FIG. 19; its sequenceis presented in FIG. 20 (SEQ ID NO:21).

Plasmid pICL contains the following features:

    ______________________________________                                        nt. 1-457   Ad5 left terminus (Sequence 1-457 of                                            human adenovirus type 5)                                          nt. 458-969 Human cytomegalovirus enhancer and                                 immediate                                                                    early promoter (Boshart et al., 1985)(from plasmid pCMVβ,                 Clontech, Palo Alto, USA)                                                    nt. 970-1204 SV40 19S exon and truncated 16/19S intron                         (from plasmid pCMVβ)                                                    nt. 1218-2987 Firefly luciferase gene (from pMLP.luc)                         nt. 3018-3131 SV40 tandem poly-adenylation signals from                        late transcript, derived from plasmid                                         pBLCAT5)                                                                     nt. 3132-5620 pUC12 backbone (derived from plasmid                             pBLCAT5)                                                                     nt. 4337-5191 β-lactamase gene (Amp-resistence gene,                      reverse orientation)                                                       ______________________________________                                    

Plasmid pICLhac and pICLhaw

Plasmids pICLhac and pICLhaw were derived from plasmid pICL by digestionof the latter plasmid with the restriction enzyme Asp718. The linearizedplasmid was treated with Calf-Intestine Alkaline Phosphatase to removethe 51 phosphate groups. The partially complementary syntheticsingle-stranded oligonucleotide HP/asp1 (SEQ ID NO:17) and HP/asp2 (SEQID NO:18) were annealed and phosphorylated on their 5' ends usingT4-polynucleotide kinase.

The phosphorylated double-stranded oligomers were mixed with thedephosporylated pICL fragment and ligated. Clones containing a singlecopy of the synthetic oligonucleotide inserted into the plasmid wereisolated and characterized using restriction enzyme digests. Insertionof the oligonucleotide into the Asp718 site will at one junctionrecreate an Asp718 recognition site, whereas at the other junction therecognition site will be disrupted. The orientation and the integrity ofthe inserted oligonucleotide was verified in selected clones by sequenceanalyses. A clone containing the oligonucleotide in the correctorientation (the Asp718 site close to the 3205 EcoRI site) was denotedpICLhac. A clone with the oligonucleotide in the reverse orientation(the Asp178 site close to the SV40 derived poly signal) was designatedpICLhaw. Plasmids pICLhac and pICLhaw are represented in FIGS. 16 and17.

Plasmid pICI was created from plasmid pICL by insertion of theSalI-SgrAI fragment from pICL, containing the Ad5-ITR into the Asp718site of pICL. The 194 bp SalI-SgrAI fragment was isolated for pICL, andthe cohesive ends were converted to blunt ends using E. coli DNApoolymerase I (Klenow fragment) and dNTP's. The Asp718 cohesive endswere converted to blunt ends by treatment with mungbean nuclease. Byligation clones were generated that contain the ITR in the Asp718 siteof plasmid pICL. A clone that contained the ITR fragment in the correctorientation was designated pICLI (FIG. 18). Generation of adenovirusAd-CMV-hcTK. Recombinant adenovirus was constructed according to themethod described in Patent application 95202213. Two components arerequired to generate a recombinant adenovirus. First, an adaptor-plasmidcontaining the left terminus of the adenovirus genome containing the ITRand the packaging signal, an expression cassette with the gene ofinterest, and a portion of the adenovirus genome which can be used forhomologous recombination. In addition, adenovirus DNA is needed forrecombination with the aforementioned adaptor plasmid. In the case ofAd-CMV-hcTK, the plasmid PCMV.TK was used as a basis. This plasmidcontains nt. 1-455 of the adenovirus type 5 genome, nt. 456-1204 derivedfrom pCMVβ (Clontech, the PstI-StuI fragment that contains the CMVenhancer promoter and the 16S/19S intron from Simian Virus 40), theHerpes Simplex Virus thymidine kinase gene (described in Patentapplication 95202213), the SV40-derived polyadenylation signal (nt.2533-2668 of the SV40 sequence), followed by the BglII-ScaI fragment ofAd5 (nt. 3328-6092 of the Ad5 sequence). These fragments are present ina pMLP10-derived (Levrero et al., 1991) backbone. To generate plasmidpAD-CMVhc-TK, plasmid pCMV.TK was digested with ClaI (the uniqueClaI-site is located just upstream of the TK open reading frame) anddephosphorylated with Calf-Intestine Alkaline Phosphate. To generate ahairpin-structure, the synthetic oligonucleotides HP/Clal (SEQ ID NO:19)HP/Cla² (SEQ ID NO:10) were annealed and phosphorylated on their 5'-OHgroups with T4-polynucleotide kinase and ATP. The double-strandedoligonucleotide was ligated with the linearized vector fragment and usedto transform E. coli strain "Sure". Insertion of the oligonucleotideinto the ClaI site will disrupt the ClaI recognition sites. In theoligonucleotide contains a new ClaI site near one of its termini. Inselected clones, the orientation and the integrity of the insertedoligonucleotide was verified by sequence analyses. A clone containingthe oligonucleotide in the correct orientation (the ClaI site at the ITRsite) was denoted pAd-CMV-hcTK. This plasmid was co-transfected withClaI digested wild-type Adenovirus-type5 DNA into 911 cells. Arecombinant adenovirus in which the CMV-hcTK expression cassettereplaces the E1 sequences was isolated and propagated using standardprocedures.

To study whether the hairpin can be used as a primer for reverse strandsynthesis on the displaced strand after replication had started at theITR. The plasmid pICLhac is introduced into 911 cells (human embryonicretinoblasts transformed with the adenovirus E1 region). The plasmidpICLhaw serves as a control, which contains the oligonucleotide pairHP/asp 1 (SEQ ID NO:17) and 2 (SEQ ID NO:18) in the reverse orientationbut is further completely identical to plasmid pICLhac. Also included inthese studies are plasmids pICLI and pICL. In the plasmid pICLI thehairpin is replaced by an adenovirus ITR. Plasmid pICL contains neithera hairpin nor an ITR sequence. These plasmids serve as controls todetermine the efficiency of replication by virtue of theterminal-hairpin structure. To provide the viral products other than theE1 proteins (these are produced by the 911 cells) required for DNAreplication the cultures are infected with the virus IG.Ad.MLPI.TK aftertransfection. Several parameters are being studies to demonstrate properreplication of the transfected DNA molecules. First, DNA extracted fromthe cell cultures transfected with aforementioned plasmids and infectedwith IG.Ad.MLPI.TK virus is being analyzed by Southern blotting for thepresence of the expected replication intermediates, as well as for thepresence of the duplicated genomes. Furthermore, from the transfectedand IG.Ad.MLPI.TK infected cell populations virus is isolated, that iscapable to transfer and express a luciferase marker gene into luciferasenegative cells.

Plasmid DNA of plasmids pIChac.pIChaw, pICI and pICL have been digestedwith restriction endonuclease SalI and treated with mungbean nuclease toremove the 4 nucleotide single-stranded extension of the resulting DNAfragment. In this manner a natural adenovirus 5'ITR terminus on the DNAfragment is created. Subsequently, both the pICLhac and pICLhaw plasmidswere digested with restriction endonuclease Asp718 to generate theterminus capable of forming a hairpin structure. The digested plasmidsare introduced into 911 cells, using the standard calcium phosphateco-precipitation technique, four dishes for each plasmid. During thetransfection, for each plasmid two of the cultures are infected with theIG.Ad.MLPI.TK virus using 5 infectious IG.Ad.MLPI.TK particles per cell.At twenty-hours post-transfection and fort hours post-transfection oneAd.tk-virus-infected and one uninfected culture are used to isolatesmall molecular-weight DNA using the procedure devised by Hirt. Aliquotsof isolated DNA are used for Southern analysis. After digestion of thesamples with restriction endonuclease EcoRI using the luciferase gene asa probe a hybridizing fragment of approx. 2.6 kb is detected only in thesamples from the adenovirus infected cells transfected with plasmidpIClhac. The size of this fragment is consistent with the anticipatedduplication of the luciferase marker gene. This supports the conclusionsthat the inserted hairpin is capable to serve as a primer for reversestrand synthesis. The hybridizing fragment is absent if theIG.Ad.MLPI.TK virus is omitted, or if the hairpin oligonucleotide hasbeen inserted in the reverse orientation.

The restriction endonuclease DpnI recognizes the tetranucleotidesequence 5'-GATC-3, but cleaves only methylated DNA, (that is, only(plasmid) DNA propagated in, and derived, from E. coli, not DNA that hasbeen replicated in mammalian cells). The restriction endonuclease MboIrecognizes the same sequences, but cleaves only unmethylated DNA (viz.DNA propagated in mammalian cells). DNA samples isolated from thetransfected cells are incubated with MboI and DpnI and analysed withSouthern blots. These results demonstrate that only in the cellstransfected with the pICLhac and the pICLI plasmids large DpnI-resistantfragments are present, that are absent in the MboI treated samples.These data demonstrate that only after transfection of plasmids pICLIand pICLhac replication and duplication of the fragments occur.

These data demonstrate that in -adenovirus-infected cells linear DNAfragments that have on one terminus an adenovirus-derived invertedterminal repeat (ITR) and at the other terminus a nucleotide sequencethat can anneal to sequences on the same strand, when present insingle-stranded form thereby generate a hairpin structure, and will beconverted to structures that have inverted terminal repeat sequences onboth ends. The resulting DNA molecules will replicate by the samemechanism as the wild type adenovirus genomes.

1.2 Demonstration that the DNA molecules that contain a luciferasemarker gene, a single copy of the ITR, the encapsidation signal and asynthetic DNA sequence, that is capable of forming a hairpin structure,are sufficient to generate DNA molecules that can be encapsidated intovirions.

To demonstrate that the above DNA molecules containing two copies of theCMV-luc marker gene can be encapsidated into virions, virus is harvestedfrom the remaining two cultures via three cycles of freeze-thaw crushingand is used to infect murine fibroblasats. Forty-eight hours afterinfection the infected cells are assayed for luciferase activity. Toexclude the possibility that the luciferase activity has been induced bytransfer of free DNA, rather than via virus particles, virus stocks aretreated with DNaseI to remove DNA contaminants. Furthermore, as anadditional control, aliquots of the virus stocks are incubated for 60minutes at 56° C. The heat treatment will not affect the contaminatingDNA, but will inactivate the viruses. Significant luciferase activity isonly found in the cells after infection with the virus stocks derivedfrom IG.Ad.MLPI.TK-infected cells transfected with the pICLhc and thepICLI plasmids. Neither in the non-infected cells, nor in the infectedcells transfected with the pIClhw and pICL significant luciferaseactivity can be demonstrated. Heat inactivation, but not DNaseItreatment, completely eliminates luciferase expression, demonstratingthat adenovirus particles, and not free (contaminating) DNA fragmentsare responsible for transfer of the luciferase reporter gene.

These results demonstrate that these small viral genomes can beencapsidated into adenovirus particles and suggest that the ITR and theencapsidation signal are sufficient for encapsidation of linear DNAfragments into adenovirus particles. These adenovirus particles can beused for efficient gene transfer. When introduced into cells thatcontains and express at least part of the adenovirus genes (viz. E1, E2,E4, and L, and VA), recombinant DNA molecules that consist of at leastone ITR, at least part of the encapsidation signal as well as asynthetic DNA sequence, that is capable of forming a hairpin structure,have the intrinsic capacity to autonomously generate recombinant genomeswhich can be encapsidated into virions. Such genomes and vector systemcan be used for gene transfer.

1.3 Demonstration that DNA molecules which contain nucleotides3510-35953 (viz. 9.7-100 map units) of the adenovirus type 5 genome(thus lack the E1 protein-coding regions, the right-hand ITR and theencapsidation sequences) and a terminal DNA sequence that iscomplementary to a portion of the same strand of the DNA molecule whenpresent in single-stranded form other than the ITR, and as a result iscapable of forming a hairpin structure, can replicate in 911 cells.

In order to develop a replicating DNA molecule that can provide theadenovirus products required to allow the above mentioned ILChac vectorgenome and alike minimal adenovectors to be encapsidated into adenovirusparticles by helper cells, the Ad-CMV-hcTK adenoviral vector has beendeveloped. Between the CMV enhancer/promoter region and the thymidinekinase gene the annealed oligonucleotide pair HP/cla 1 (SEQ ID NO:19)and 2 (SEQ ID NO:20) is inserted. The vector Ad-CMV-hcTK can bepropagated and produced in 911 cell using standard procedures. Thisvector is grown and propagated exclusively as a source of DNA used fortransfection. DNA of the adenovirus Ad-CMV-hcTK is isolated from virusparticles that had been purified using CsCl density-gradientcentrifugation by standard techniques. The virus DNA has been digestedwith restriction endonuclease ClaI. The digested DNA is size-fractionateon an 0.7% agarose gel and the large fragment is isolated and used forfurther experiments. Cultures of 911 cells are transfected largeClaI-fragment of the Ad-CMV-hcTK DNA using the standard calciumphosphate co-precipitation technique. Much like in the previousexperiments with plasmid pICLhac, the AD-CMV-hc will replicate startingat the right-hand ITR. Once the 1-strand is displaced, a hairpin can beformed at the left-hand terminus of the fragment. This facilitates theDNA polymerase to elongate the chain towards the right-hand-side. Theprocess will proceed until the displaced strand is completely convertedto its double-stranded form. Finally, the right-hand ITR will berecreated, and in this location the normal adenovirusreplication-initiation and elongation will occur. Note that thepolymerase will read through the hairpin, thereby duplicating themolecule. The input DNA molecule of 33250 bp, that had on one side anadenovirus ITR sequence and at the other side a DNA sequence that hadthe capacity to form a hairpin structure, has now been duplicated, in away that both ends containing an ITR sequence. The resulting DNAmolecule will consist of a palindromic structure of approximately 66500bp.

This structure can be detected in low-molecular weight DNA extractedfrom the transfected cells using Southern analysis. The palindromicnature of the DNA fragment can be demonstrated by digestion of thelow-molecular weight DNA with suitable restriction endonucleases andSouthern blotting with the HSV-TK gene as the probe. This molecule canreplicate itself in the transfected cells by virtue of the adenovirusgene products that are present in the cells. In part, the adenovirusgenes are expressed from templates that are integrated in the genome ofthe target cells (viz. the E1 gene products), the other genes reside inthe replicating DNA fragment itself. Note however, that this linear DNAfragment cannot be encapsidated into virions. Not only does it lack allthe DNA sequences required for encapsidation, but also is its size muchtoo large to be encapsidated.

1.4 Demonstration that DNA molecules which contain nucleotides3503-35953 (viz. 9.7-100 map units) of the adenovirus type 5 genome(thus lack the E1 protein-coding regions, the right-hand ITR and theencapsidation sequences) and a terminal DNA sequence that iscomplementary to a portion the same strand of the DNA molecule otherthan the ITR, and as a result is capable of forming a hairpin structure,can replicate in 911 cells and can provide the helper functions requiredto encapsidate the pICLI and pICLhac derived DNA fragments.

The next series of experiments aim to demonstrate that the DNA moleculedescribed in part 1.3 could be used to encapsidate the minimaladenovectors described in part 1.1 and 1.2.

In the experiments the large fragment isolated after endonucleaseClaI-digestion of Ad-CMV-hcTK DNA is introduced into 911 cells (conformthe experiments described in part 1.3) together with endonuclease SalI,mungbean nuclease, endonuclease Asp718-treated plasmid pICLhac, or as acontrol similarly treated plasmid pICLhaw. After 48 hours virus isisolated by freeze-thaw crushing of the transfected cell population. Thevirus-preparation is treated with DNaseI to remove contaminating freeDNA. The virus is used subsequently to infect Rat2 fibroblasts.Forty-eight hours post infection the cells are assayed for luciferaseactivity. Only in the cells infected with virus isolated from the cellstransfected with the pICLhac plasmid, and not with the pICLhaw plasmid,significant luciferase activity can be demonstrated. Heat inactivationof the virus prior to infection completely abolishes the luciferaseactivity, indicating that the luciferase gene is transferred by a viralparticle. Infection of 911 cell with the virus stock did not result inany cytopathological effects, demonstrating that the pICLhac is producedwithout any infectious helper virus that can be propagated on 911 cells.These results demonstrate that the proposed method can be used toproduce stocks of minimal-adenoviral vectors, that are completely devoidof infectious helper viruses that are able to replicate autonomously onadenovirus-transformed human cells or on non-adenovirus transformedhuman cells.

Besides the system described in this application, another approach forthe generation of minimal adenovirus vectors has been disclosed in WO94/12649. The method described in WO 94/12649 exploits the function ofthe protein IX for the packaging of minimal adenovirus vectors (PseudoAdenoviral Vectors (PAV) in the terminology of WO 94/12649). PAVs areproduced by cloning an expression plasmid with the gene of interestbetween the left-hand (including the sequences required forencapsidation) and the right-hand adenoviral ITRs. The PAV is propagatedin the presence of a helper virus. Encapsidation of the PAV is preferredcompared the helper virus because the helper virus is partiallydefective for packaging. (Either by virtue of mutations in the packagingsignal or by virtue of its size (virus genomes greater than 37.5 kbpackage inefficiently). In addition, the authors propose that in theabsence of the protein IX gene the PAV will be preferentially packaged.However, neither of these mechanisms appear to be sufficientlyrestrictive to allow packaging of only PAVs/minimal vectors. Themutations proposed in the packaging signal diminish packaging, but donot provide an absolute block as the same packaging-activity is requiredto propagate the helper virus. Also neither an increase in the size ofthe helper virus nor the mutation of the protein IX gene will ensurethat PAV is packaged exclusively. Thus, the method described in WO94/12649 is unlikely to be useful for the production of helper-freestocks of minimal adenovirus vectors/PAVs.

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Yang, Y., Nunes, F. A. Berencsi, K., Gonczol, E., Engelhardt, J. F., andWilson, J. M. (1994b): Inactivation of E2A in recombinant adenovirusesimproves the prospect for gene therapy in cystic fibrosis. Nat Genet 7,362-9.

Zantema, A., Fransen, J. A. M., Davis-Olivier, A., Ramaekers, F. C. S.,Vooijs, G. P., Deleys, B., and Eb. A. J. v. d. (1985). Localization ofthe E1B proteins of adenovirus 5 in transformed cells, as revealed byinteraction with monoclonal antibodies. Virology 142, 44-58.

                                      TABLE I                                     __________________________________________________________________________     -                                                                            Primers used for PCR amplification of DNA fragments used                        for generation of constructs described in this patent                         application.                                                                __________________________________________________________________________       -                                                                          Ea-1 (SEQ ID NO:1)                                                                           CGTGTAGTGTATTTATACCCG                                                                             PCR amplification Ad5 nt459 ->                                                  - Ea-2 (SEQ ID NO:2) TCGTCACTGGGTGGAA                                       AGCCA PCR amplification Ad5 nt960 <-                                            - Ea-3 (SEQ ID NO:3) TACCCGCCGTCCTAAA                                       ATGGC nt1284-1304 of Ad5 genome                                                 - Ea-5 (SEQ ID NO:4) TGGACTTGAGCTGTAA                                       ACGC nt1514-1533 of Ad5 genome                                                  - Ep-2 (SEQ ID NO:5) GCCTCCATGGAGGTCA                                       GATGT nt1721-1702 of Ad5:                       introduction of NcoI site                                                   - Eb-1 (SEQ ID NO:6) GCTTGAGCCCGAGACATGTC nt3269-3289 of Ad5 genome                                             - Eb-2 (SEQ ID NO:7) CCCCTCGAGCTCAATC                                       TGTATCTT nt3508-3496 of Ad5 genome:                                               introduction of XhoI site                 - SV40-1 (SEQ ID NO:8) GGGGGATCCGAACTTGTTTATTGCAGC Introduction BamHI                                         site                                            (nt2182-2199 of pMLP.TK)                                                      adaption of ecombinant                                                        adenoviruses                                                                - SV40-2 (SEQ ID NO:9) GGGAGATCTAGACATGATAAGATAC Introduction BglII                                           site                                            (nt2312-2297 of pMLP.TK)                                                    - Ad5-1 (SEQ ID NO:10) GGGAGATCTGTACTGAAATGTGTGGGC Introduction BglII                                         site                                            (nt2496-2514 of pMLP.TK)                                                    - Ad5-2 (SEQ ID NO:11) GGAGGCTGCAGTCTCCAACGGCGT nt2779-2756 of PMLP.TK        - ITR1 (SEQ ID NO:12) GGGGGATCCTCAAATCGTCACTTCCGT nt35737-35757 of Ad5          (introduction of BamHI site)                                                - ITR2 (SEQ ID NO:13) GGGGTCTAGACATCATCAATAATATAC nt35935-35919 of Ad5          (introduction of XbaI site)                                                 -                                                                          PCR primers sets to be used to create the SalI and Asp718                       sites juxtaposed to the ITR sequences.                                      __________________________________________________________________________       -                                                                          PCR/MLP1                                                                           (SEQ ID NO:14)                                                                          GGCGAATTCGTCGACATCATCAATAATATACC                                                                  (Ad5 nt. 10-18)                               - PCR/MLP2 (SEQ ID NO:15) GGCGAATTCGGTACCATCATCAATAATATACC (Ad5 nt.                                           10-18)                                        - PCR/MLP3 (SEQ ID NO:16) CTGTGTACACCGGCGCA (Ad5 nt.200-184)                  -                                                                          Synthetic oligonucleotide pair used to generate a                               synthetic hairpin, recreates an Asp718 site at one of the                     termini if inserted in Asp718 site:                                         __________________________________________________________________________       -                                                                          HP/asp1                                                                            (SEQ ID NO:17)                                                                          5'-GTACACTGACCTAGTGCCGCCCGGGCAAAGCCCGGGCGGCACTAGGTCAG                           - HP/asp2 (SEQ ID NO:18) 5'-GTACCTGACCTAGTGCCGCCCGGGCTTTG                   CCCGGGCGGCACTAGGTCAGT                                             -                                                                          Synthetic oligonucleotide pair used to generate a                               synthetic hairpin, contains the ClaI recognition site to                      be used for hairpin formation.                                              __________________________________________________________________________       -                                                                          HP/cla1                                                                            (SEQ ID NO:19)                                                                          5'-GTACATTGACCTAGTGCCGCCCGGGCAAAGCCCGGGCGGCACTAGGTCAATCGAT        - HP/cla2 (SEQ ID NO:20) 5'-GTACATCGATTGACCTAGTGCCGCCCGGGCTTTGCCCGGGCGG                   CACTAGGTCAAT                                                   __________________________________________________________________________

                                      TABLE II                                    __________________________________________________________________________        Passage                        Producer                                     Cell number IG.Ad.CMV.lacZ IG.Ad.CMV.TK IG.Ad.MLPI.TK dl313 Mean            __________________________________________________________________________    293     6.0     5.8     24      34 17.5                                         911  8 14 34 180 59.5                                                         PER.C3 17 8 11 44 40 25.8                                                     PER.C5 15 6 17 36 200 64.7                                                    PER.C6 36 10 22 58 320 102                                                  __________________________________________________________________________     Yields × 10.sup.-8 pfu/T175 flask.                                      Yields of different recombinant adenoviruses obtained after inoculation o     adenovirus E1 packaging cell lines 293, 911, PER.C3, PER.C5 and PER.C6.       The yields are the mean of two different experiments.                         IG.Ad.CMV.lacZ and IG.Ad.CMV.TK are described in patent application EP 95     20 2213                                                                       The construction of IG.Ad.MLPI.TK is described in this patent application     Yields of virus per T80 flask were determined by plaque assay on 911          cells, as described [Fallaux, 1996 #1493                                 

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 22                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - CGTGTAGTGT ATTTATACCC G           - #                  - #                      - #21                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - TCGTCACTGG GTGGAAAGCC A           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - TACCCGCCGT CCTAAAATGG C           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - TGGACTTGAG CTGTAAACGC            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - GCCTCCATGG AGGTCAGATG T           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - GCTTGAGCCC GAGACATGTC            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - CCCCTCGAGC TCAATCTGTA TCTT          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - GGGGGATCCG AACTTGTTTA TTGCAGC          - #                  - #                 27                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - GGGAGATCTA GACATGATAA GATAC          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - GGGAGATCTG TACTGAAATG TGTGGGC          - #                  - #                 27                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - GGAGGCTGCA GTCTCCAACG GCGT          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - GGGGGATCCT CAAATCGTCA CTTCCGT          - #                  - #                 27                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - GGGGTCTAGA CATCATCAAT AATATAC          - #                  - #                 27                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - GGCGAATTCG TCGACATCAT CAATAATATA CC       - #                  - #              32                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - GGCGAATTCG GTACCATCAT CAATAATATA CC       - #                  - #              32                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - CTGTGTACAC CGGCGCA             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - GTACACTGAC CTAGTGCCGC CCGGGCAAAG CCCGGGCGGC ACTAGGTCAG  - #                  50                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - - GTACCTGACC TAGTGCCGCC CGGGCTTTGC CCGGGCGGCA CTAGGTCAGT  - #                  50                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 55 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - - GTACATTGAC CTAGTGCCGC CCGGGCAAAG CCCGGGCGGC ACTAGGTCAA TC - #GAT              55                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 55 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - - GTACATCGAT TGACCTAGTG CCGCCCGGGC TTTGCCCGGG CGGCACTAGG TC - #AAT              55                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5620 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                               - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "plasmid"                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                              - - CATCATCAAT AATATACCTT ATTTTGGATT GAAGCCAATA TGATAATGAG GG -             #GGTGGAGT     60                                                                 - - TTGTGACGTG GCGCGGGGCG TGGGAACGGG GCGGGTGACG TAGTAGTGTG GC -            #GGAAGTGT    120                                                                 - - GATGTTGCAA GTGTGGCGGA ACACATGTAA GCGACGGATG TGGCAAAAGT GA -            #CGTTTTTG    180                                                                 - - GTGTGCGCCG GTGTACACAG GAAGTGACAA TTTTCGCGCG GTTTTAGGCG GA -            #TGTTGTAG    240                                                                 - - TAAATTTGGG CGTAACCGAG TAAGATTTGG CCATTTTCGC GGGAAAACTG AA -            #TAAGAGGA    300                                                                 - - AGTGAAATCT GAATAATTTT GTGTTACTCA TAGCGCGTAA TATTTGTCTA GG -            #GCCGCGGG    360                                                                 - - GACTTTGACC GTTTACGTGG AGACTCGCCC AGGTGTTTTT CTCAGGTGTT TT -            #CCGCGTTC    420                                                                 - - CGGGTCAAAG TTGGCGTTTT ATTATTATAG TCAGGGGCTG CAGGTCGTTA CA -            #TAACTTAC    480                                                                 - - GGTAAATGGC CCGCCTGGCT GACCGCCCAA CGACCCCCGC CCATTGACGT CA -            #ATAATGAC    540                                                                 - - GTATGTTCCC ATAGTAACGC CAATAGGGAC TTTCCATTGA CGTCAATGGG TG -            #GAGTATTT    600                                                                 - - ACGGTAAACT GCCCACTTGG CAGTACATCA AGTGTATCAT ATGCCAAGTA CG -            #CCCCCTAT    660                                                                 - - TGACGTCAAT GACGGTAAAT GGCCCGCCTG GCATTATGCC CAGTACATGA CC -            #TTATGGGA    720                                                                 - - CTTTCCTACT TGGCAGTACA TCTACGTATT AGTCATCGCT ATTACCATGG TG -            #ATGCGGTT    780                                                                 - - TTGGCAGTAC ATCAATGGGC GTGGATAGCG GTTTGACTCA CGGGGATTTC CA -            #AGTCTCCA    840                                                                 - - CCCCATTGAC GTCAATGGGA GTTTGTTTTG GCACCAAAAT CAACGGGACT TT -            #CCAAAATG    900                                                                 - - TCGTAACAAC TCCGCCCCAT TGACGCAAAT GGGCGGTAGG CGTGTACGGT GG -            #GAGGTCTA    960                                                                 - - TATAAGCAGA GCTCGTTTAG TGAACCGTCA GATCGCCTGG AGACGCCATC CA -            #CGCTGTTT   1020                                                                 - - TGACCTCCAT AGAAGACACC GGGACCGATC CAGCCTCCGG ACTCTAGAGG AT -            #CCGGTACT   1080                                                                 - - CGAGGAACTG AAAAACCAGA AAGTTAACTG GTAAGTTTAG TCTTTTTGTC TT -            #TTATTTCA   1140                                                                 - - GGTCCCGGAT CCGGTGGTGG TGCAAATCAA AGAACTGCTC CTCAGTGGAT GT -            #TGCCTTTA   1200                                                                 - - CTTCTAGTAT CAAGCTTGAA TTCCTTTGTG TTACATTCTT GAATGTCGCT CG -            #CAGTGACA   1260                                                                 - - TTAGCATTCC GGTACTGTTG GTAAAATGGA AGACGCCAAA AACATAAAGA AA -            #GGCCCGGC   1320                                                                 - - GCCATTCTAT CCTCTAGAGG ATGGAACCGC TGGAGAGCAA CTGCATAAGG CT -            #ATGAAGAA   1380                                                                 - - ATACGCCCTG GTTCCTGGAA CAATTGCTTT TACAGATGCA CATATCGAGG TG -            #AACATCAC   1440                                                                 - - GTACGCGGAA TACTTCGAAA TGTCCGTTCG GTTGGCAGAA GCTATGAAAC GA -            #TATGGGCT   1500                                                                 - - GAATACAAAT CACAGAATCG TCGTATGCAG TGAAAACTCT CTTCAATTCT TT -            #ATGCCGGT   1560                                                                 - - GTTGGGCGCG TTATTTATCG GAGTTGCAGT TGCGCCCGCG AACGACATTT AT -            #AATGAACG   1620                                                                 - - TGAATTGCTC AACAGTATGA ACATTTCGCA GCCTACCGTA GTGTTTGTTT CC -            #AAAAAGGG   1680                                                                 - - GTTGCAAAAA ATTTTGAACG TGCAAAAAAA ATTACCAATA ATCCAGAAAA TT -            #ATTATCAT   1740                                                                 - - GGATTCTAAA ACGGATTACC AGGGATTTCA GTCGATGTAC ACGTTCGTCA CA -            #TCTCATCT   1800                                                                 - - ACCTCCCGGT TTTAATGAAT ACGATTTTGT ACCAGAGTCC TTTGATCGTG AC -            #AAAACAAT   1860                                                                 - - TGCACTGATA ATGAATTCCT CTGGATCTAC TGGGTTACCT AAGGGTGTGG CC -            #CTTCCGCA   1920                                                                 - - TAGAACTGCC TGCGTCAGAT TCTCGCATGC CAGAGATCCT ATTTTTGGCA AT -            #CAAATCAT   1980                                                                 - - TCCGGATACT GCGATTTTAA GTGTTGTTCC ATTCCATCAC GGTTTTGGAA TG -            #TTTACTAC   2040                                                                 - - ACTCGGATAT TTGATATGTG GATTTCGAGT CGTCTTAATG TATAGATTTG AA -            #GAAGAGCT   2100                                                                 - - GTTTTTACGA TCCCTTCAGG ATTACAAAAT TCAAAGTGCG TTGCTAGTAC CA -            #ACCCTATT   2160                                                                 - - TTCATTCTTC GCCAAAAGCA CTCTGATTGA CAAATACGAT TTATCTAATT TA -            #CACGAAAT   2220                                                                 - - TGCTTCTGGG GGCGCACCTC TTTCGAAAGA AGTCGGGGAA GCGGTTGCAA AA -            #CGCTTCCA   2280                                                                 - - TCTTCCAGGG ATACGACAAG GATATGGGCT CACTGAGACT ACATCAGCTA TT -            #CTGATTAC   2340                                                                 - - ACCCGAGGGG GATGATAAAC CGGGCGCGGT CGGTAAAGTT GTTCCATTTT TT -            #GAAGCGAA   2400                                                                 - - GGTTGTGGAT CTGGATACCG GGAAAACGCT GGGCGTTAAT CAGAGAGGCG AA -            #TTATGTGT   2460                                                                 - - CAGAGGACCT ATGATTATGT CCGGTTATGT AAACAATCCG GAAGCGACCA AC -            #GCCTTGAT   2520                                                                 - - TGACAAGGAT GGATGGCTAC ATTCTGGAGA CATAGCTTAC TGGGACGAAG AC -            #GAACACTT   2580                                                                 - - CTTCATAGTT GACCGCTTGA AGTCTTTAAT TAAATACAAA GGATATCAGG TG -            #GCCCCCGC   2640                                                                 - - TGAATTGGAA TCGATATTGT TACAACACCC CAACATCTTC GACGCGGGCG TG -            #GCAGGTCT   2700                                                                 - - TCCCGACGAT GACGCCGGTG AACTTCCCGC CGCCGTTGTT GTTTTGGAGC AC -            #GGAAAGAC   2760                                                                 - - GATGACGGAA AAAGAGATCG TGGATTACGT CGCCAGTCAA GTAACAACCG CG -            #AAAAAGTT   2820                                                                 - - GCGCGGAGGA GTTGTGTTTG TGGACGAAGT ACCGAAAGGT CTTACCGGAA AA -            #CTCGACGC   2880                                                                 - - AAGAAAAATC AGAGAGATCC TCATAAAGGC CAAGAAGGGC GGAAAGTCCA AA -            #TTGTAAAA   2940                                                                 - - TGTAACTGTA TTCAGCGATG ACGAAATTCT TAGCTATTGT AATGGGGGAT CC -            #CCAACTTG   3000                                                                 - - TTTATTGCAG CTTATAATGG TTACAAATAA AGCAATAGCA TCACAAATTT CA -            #CAAATAAA   3060                                                                 - - GCATTTTTTT CACTGCATTC TAGTTGTGGT TTGTCCAAAC TCATCAATGT AT -            #CTTATCAT   3120                                                                 - - GTCTGGATCG GATCGATCCC CGGGTACCGA GCTCGAATTC GTAATCATGG TC -            #ATAGCTGT   3180                                                                 - - TTCCTGTGTG AAATTGTTAT CCGCTCACAA TTCCACACAA CATACGAGCC GG -            #AAGCATAA   3240                                                                 - - AGTGTAAAGC CTGGGGTGCC TAATGAGTGA GCTAACTCAC ATTAATTGCG TT -            #GCGCTCAC   3300                                                                 - - TGCCCGCTTT CCAGTCGGGA AACCTGTCGT GCCAGCTGCA TTAATGAATC GG -            #CCAACGCG   3360                                                                 - - CGGGGAGAGG CGGTTTGCGT ATTGGGCGCT CTTCCGCTTC CTCGCTCACT GA -            #CTCGCTGC   3420                                                                 - - GCTCGGTCGT TCGGCTGCGG CGAGCGGTAT CAGCTCACTC AAAGGCGGTA AT -            #ACGGTTAT   3480                                                                 - - CCACAGAATC AGGGGATAAC GCAGGAAAGA ACATGTGAGC AAAAGGCCAG CA -            #AAAGGCCA   3540                                                                 - - GGAACCGTAA AAAGGCCGCG TTGCTGGCGT TTTTCCATAG GCTCCGCCCC CC -            #TGACGAGC   3600                                                                 - - ATCACAAAAA TCGACGCTCA AGTCAGAGGT GGCGAAACCC GACAGGACTA TA -            #AAGATACC   3660                                                                 - - AGGCGTTTCC CCCTGGAAGC TCCCTCGTGC GCTCTCCTGT TCCGACCCTG CC -            #GCTTACCG   3720                                                                 - - GATACCTGTC CGCCTTTCTC CCTTCGGGAA GCGTGGCGCT TTCTCATAGC TC -            #ACGCTGTA   3780                                                                 - - GGTATCTCAG TTCGGTGTAG GTCGTTCGCT CCAAGCTGGG CTGTGTGCAC GA -            #ACCCCCCG   3840                                                                 - - TTCAGCCCGA CCGCTGCGCC TTATCCGGTA ACTATCGTCT TGAGTCCAAC CC -            #GGTAAGAC   3900                                                                 - - ACGACTTATC GCCACTGGCA GCAGCCACTG GTAACAGGAT TAGCAGAGCG AG -            #GTATGTAG   3960                                                                 - - GCGGTGCTAC AGAGTTCTTG AAGTGGTGGC CTAACTACGG CTACACTAGA AG -            #GACAGTAT   4020                                                                 - - TTGGTATCTG CGCTCTGCTG AAGCCAGTTA CCTTCGGAAA AAGAGTTGGT AG -            #CTCTTGAT   4080                                                                 - - CCGGCAAACA AACCACCGCT GGTAGCGGTG GTTTTTTTGT TTGCAAGCAG CA -            #GATTACGC   4140                                                                 - - GCAGAAAAAA AGGATCTCAA GAAGATCCTT TGATCTTTTC TACGGGGTCT GA -            #CGCTCAGT   4200                                                                 - - GGAACGAAAA CTCACGTTAA GGGATTTTGG TCATGAGATT ATCAAAAAGG AT -            #CTTCACCT   4260                                                                 - - AGATCCTTTT AAATTAAAAA TGAAGTTTTA AATCAATCTA AAGTATATAT GA -            #GTAAACTT   4320                                                                 - - GGTCTGACAG TTACCAATGC TTAATCAGTG AGGCACCTAT CTCAGCGATC TG -            #TCTATTTC   4380                                                                 - - GTTCATCCAT AGTTGCCTGA CTCCCCGTCG TGTAGATAAC TACGATACGG GA -            #GGGCTTAC   4440                                                                 - - CATCTGGCCC CAGTGCTGCA ATGATACCGC GAGACCCACG CTCACCGGCT CC -            #AGATTTAT   4500                                                                 - - CAGCAATAAA CCAGCCAGCC GGAAGGGCCG AGCGCAGAAG TGGTCCTGCA AC -            #TTTATCCG   4560                                                                 - - CCTCCATCCA GTCTATTAAT TGTTTGCCGG AAGCTAGAGT AAGTAGTTCG CC -            #AGTTAATA   4620                                                                 - - GTTTGCGCAA CGTTGTTGCC ATTGCTACAG GCATCGTGGT GTCACGCTCG TC -            #GTTTGGTA   4680                                                                 - - TGGCTTCATT CAGCTCCGGT TCCCAACGAT CAAGGCGAGT TACATGATCC CC -            #CATGTTGT   4740                                                                 - - GCAAAAAAGC GGTTAGCTCC TTCGGTCCTC CGATCGTTGT CAGAAGTAAG TT -            #GGCCGCAG   4800                                                                 - - TGTTATCACT CATGGTTATG GCAGCACTGC ATAATTCTCT TACTGTCATG CC -            #ATCCGTAA   4860                                                                 - - GATGCTTTTC TGTGACTGGT GAGTACTCAA CCAAGTCATT CTGAGAATAG TG -            #TATGCGGC   4920                                                                 - - GACCGAGTTG CTCTTGCCCG GCGTCAATAC GGGATAATAC CGCGCCACAT AG -            #CAGAACTT   4980                                                                 - - TAAAAGTGCT CATCATTGGA AAACGTTCTT CGGGGCGAAA ACTCTCAAGG AT -            #CTTACCGC   5040                                                                 - - TGTTGAGATC CAGTTCGATG TAACCCACTC GTGCACCCAA CTGATCTTCA GC -            #ATCTTTTA   5100                                                                 - - CTTTCACCAG CGTTTCTGGG TGAGCAAAAA CAGGAAGGCA AAATGCCGCA AA -            #AAAGGGAA   5160                                                                 - - TAAGGGCGAC ACGGAAATGT TGAATACTCA TACTCTTCCT TTTTCAATAT TA -            #TTGAAGCA   5220                                                                 - - TTTATCAGGG TTATTGTCTC ATGAGCGGAT ACATATTTGA ATGTATTTAG AA -            #AAATAAAC   5280                                                                 - - AAATAGGGGT TCCGCGCACA TTTCCCCGAA AAGTGCCACC TGACGTCTAA GA -            #AACCATTA   5340                                                                 - - TTATCATGAC ATTAACCTAT AAAAATAGGC GTATCACGAG GCCTATGCGG TG -            #TGAAATAC   5400                                                                 - - CGCACAGATG CGTAAGGAGA AAATACCGCA TCAGGCGCCA TTCGCCATTC AG -            #GCTGCGCA   5460                                                                 - - ACTGTTGGGA AGGGCGATCG GTGCGGGCCT CTTCGCTATT ACGCCAGCTG GC -            #GAAAGGGG   5520                                                                 - - GATGTGCTGC AAGGCGATTA AGTTGGGTAA CGCCAGGGTT TTCCCAGTCA CG -            #ACGTTGTA   5580                                                                 - - AAACGACGGC CAGTGCCAAG CTTGCATGCC TGCAGGTCGA     - #                      - #  5620                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -    (iii) HYPOTHETICAL: YES                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                              - - GTACACTGAC CTAGTGCCGC CCGGGCAAAG CCCGGGCGGC ACTAG   - #                      - #45                                                                    __________________________________________________________________________

We claim:
 1. A system for producing adenovirus incapable of replicating,said system comprising:a primary cell containing a nucleic acid encodingadenoviral E1A and E1B gene products, wherein aid nucleic acid lacks agene coding for active or functional pIX; and an isolated recombinantnucleic acid molecule for transfer into said primary cell, said isolatedrecombinant nucleic acid molecule based on or derived from an adenovirusof the family Adenoviridae, and further having at least a functionalencapsidating signal, and at least one functional Inverted TerminalRepeat, said isolated recombinant nucleic acid molecule lackingoverlapping sequences with the nucleic acid of the cell, the overlappingsequences otherwise enabling homologous recombination leading toreplication competent adenovirus in said primary cell into which saidisolated recombinant nucleic acid molecule is to be transferred.
 2. Thesystem of claim 1 wherein said isolated recombinant nucleic acidmolecule is in a linear form and comprises functional Inverted TerminalRepeats at or near both termini.
 3. The system of claim 1 wherein saidisolated recombinant nucleic acid molecule comprises a nucleic acidwhich alters the range of said adenovirus as compared to a wild-typeadenovirus and wherein said primary cell is a non-human primary cell. 4.An adenoviral producer cell that does not produce replication competentadenovirus, said adenoviral producer cell comprising:one or morerecombinant nucleic acid molecules having no overlapping sequences withrespect to one another which would otherwise allow for homologousrecombination leading to replication competent adenovirus in saidadenoviral producer cell, and wherein said adenoviral producer cellcomprises a nucleic acid encoding adenoviral E1A and E1B gene products,said nucleic acid lacking a gene coding for functional or active pIX. 5.An adenoviral producer cell that does not produce replication competentadenovirus, said adenoviral producer cell comprising:one or morerecombinant nucleic acid molecules having no overlapping sequences withrespect to one another which would otherwise allow for homologousrecombination leading to replication competent adenovirus in saidadenoviral producer cell, and wherein said adenoviral producer cellcomprises a nucleic acid encoding adenoviral E1 and E1B gene products,said nucleic acid lacking a gene coding for functional or active pIX,and E2A gene product under the control of an inducible promoter.
 6. Anadenoviral producer cell that does not produce replication competentadenovirus, said adenoviral producer cell comprising:one or morerecombinant nucleic acid molecules having no overlapping sequences withrespect to one another which would otherwise allow for homologousrecombination leading to replication competent adenovirus in saidadenoviral producer cell, said adenoviral producer cell comprises anucleic acid encoding adenoviral E1 and E2A gene products, said nucleicacid lacking a gene coding for functional or active pIX, and whereinsaid E2A gene product encodes a temperature sensitive mutation.
 7. Theproducer cell according to claim 4, wherein any one or more of saidrecombinant nucleic acid molecules thereof further comprises a markergene.
 8. The producer cell according to claim 7, wherein the marker geneis under control of an E1A adenoviral gene product responsive promoter.9. The producer cell according to claim 4 or 5, which is a diploid cell.10. The producer cell according to claim 4 or 5, which is of non-humanorigin.
 11. The producer cell according to claim 3 which is of monkeyorigin.
 12. The system of claim 3 wherein said isolated recombinantnucleic acid molecule is DNA.
 13. The adenoviral producer cell of claim4 or 5 wherein at least one of said one or more recombinant nucleic acidmolecules comprises at least one functional encapsidating signal and onefunctional Inverted Terminal Repeat, but lacks nucleotides 459-3510 ofthe E1 gene of adenovirus.
 14. The adenoviral producer cell of claim 4or 5 wherein at least one of said one or more recombinant nucleic acidmolecules comprises at least one functional encapsidating signal and onefunctional Inverted Terminal Repeat, but lacks nucleotides 459-1713 ofthe E1 gene of adenovirus.
 15. The producer cell according to claim 4wherein a recombinant nucleic acid molecule thereof further comprises amutated E2A adenoviral gene that encodes a temperature sensitive geneproduct.
 16. An established adenoviral producer cell that lacks a geneencoding for active or functional pIX and does not produce replicationcompetent adenovirus, said established adenoviral producer cell derivedfrom a primary cell, said established adenoviral producer cellcomprising:one or more recombinant nucleic acid molecules having nooverlapping sequences within each of the recombinant nucleic acidmolecules, wherein said adenoviral producer cell comprises DNA sequencesencoding functional adenoviral E1A and E1B gene products.
 17. Theestablished adenoviral producer cell of claim 16 wherein at least one ofsaid one or more recombinant nucleic acid molecules further comprisesDNA sequences encoding an adenoviral E2A gene product.
 18. Theestablished adenoviral producer cell of claim 16 wherein said DNAsequence encoding an adenoviral E2A gene product is selected from thegroup consisting of a DNA sequence encoding the wild-type E2A geneoperably linked to an inducible promoter and a DNA sequence encoding atemperature sensitive 125 mutation.
 19. The producer cell of claim 6wherein said E2A gene product is under the control of an induciblepromoter.
 20. The system of claim 1 wherein said primary cell comprisesa nucleic acid encoding a mutated E2 gene product which alters the hostrange of said adenovirus as compared to a wild-type adenovirus.